Wheat malt seedling functional dietary fiber as well as preparation method and application thereof

Abstract

The invention discloses wheat malt seedling functional dietary fibers as well as a preparation method and application of the wheat malt seedling functional dietary fibers. The soluble dietary fiber and the insoluble dietary fiber of the wheat malt seedlings are extracted by adopting a wall breaking-extraction mode of synergy of biological enzyme and free radicals. The wheat malt seedling soluble dietary fibers can prolong the life of caenorhabditis elegans in an oxidative stress state, improve the antioxidant enzyme activity and improve the anti-apoptosis capability; the intestinal flora of the obese C57BL / 6J mouse induced by the high-fat feed is regulated; the wheat malt seedling insoluble dietary fiber has better binding water capacity, water-holding capacity and cholesterol and sodium cholate adsorption capacity, and can be used as a functional food additive for improving the texture and nutritional characteristics of food; and the wheat malt seedlings can also be used for assisting in reducing serum cholesterol level, preventing arteriosclerosis, promoting defecation, preventing constipation, colorectal cancer and the like in the field of health medical treatment, so that high-quality reutilization of the wheat malt seedlings is realized.

Description

(I) TECHNICAL FIELD

[0001] The present application relates to a kind of wheat sprout functional dietary fiber and its preparation method and application. (II) BACKGROUND

[0002] Wheat, as one of the three major global staple foods that have nourished human civilization for thousands of years, its traditional utilization method has long been focused on grain processing, forming flour, noodles, bread and other basic staple foods. However, under the dual driving of the progress of food science and technology and the vigorous rise of global health consumption awareness, the value chain of wheat is undergoing a profound transformation and expansion. Scientific research reveals that during the germination and early seedling stage (3-15 days after germination), the internal metabolic engine of wheat seed is strongly activated, and a complex biochemical cascade reaction occurs: macromolecular substances are efficiently decomposed and transformed into easily absorbed small molecule nutrients; at the same time, the content and bioavailability of bioactive substances such as dietary fiber (especially soluble dietary fiber SDF) and phenols are significantly improved. This dynamic biological transformation process endows wheat sprouts with unique nutritional and functional properties beyond the grain itself, making it contain excellent antioxidant capacity, potential anti-aging effect and potential potential for improving intestinal health, which meets the needs of modern consumers for natural and functional foods.

[0003] The global functional food and beverage market is showing an explosive growth trend. Consumers have strong demand for "clean label", "natural source" and "preventive health" products, driving the industry to continuously seek new health raw materials with scientific backing and consumer appeal. Wheat sprouts, with their affinity to staple food culture, healthiness of "whole grain / germination", sustainability and cost advantage, have become an ideal carrier for developing high-value functional foods. However, the current development of wheat sprouts faces the following problems: there is little research on the active ingredients of wheat sprouts, and there are few reports on the exact active ingredients in wheat sprouts. The value of key active components in wheat sprouts has not been systematically analyzed; existing products are mostly limited to direct consumption of sprouts, and no high-value industrial chain has been formed.

[0004] Therefore, in the case that the active ingredients in the tracked wheat sprouts are unknown, by using the solvent extraction method of classical prescriptions in the motherland, and combining with the pharmacological test of active oxygen and active nitrogen free radical scavenging ability, the wheat sprout powder is extracted with different solvents to determine the effective components, and then the components with the most concentrated effective components are gradually divided and tracked. This method can ensure that the active ingredients in wheat sprouts are not lost. That is, using germination period wheat as raw material, through the whole-chain development path of "active ingredient dynamic tracking → targeted green extraction → anti-aging function evaluation → terminal product creation", the transformation and upgrading of wheat industry from traditional staple food to high-value health food is promoted. (III) SUMMARY

[0005] The application aims to provide a wheat sprout functional dietary fiber and a preparation method and application thereof, and the application takes ultra-micro wheat sprout powder as a raw material, first adopts hot water extraction to extract as many as possible hydrophilic components such as saccharides, amino acids, peptides, proteins and organic acids in the wheat sprout, then uses strong penetration of ethanol to extract as many as possible components such as glycosides, alkaloid salts, polyols, tannins and substances with small polarity such as aglycone, alkaloids, fatty oils and sterols in the wheat sprout, combines two kinds of extraction liquids (wheat sprout crude extract) and then adopts organic solvent step extraction according to solvent polarity increasing method to classify the wheat sprout crude extract into four different components. The scavenging capacity of active oxygen (OH) and active nitrogen (ABTS) free radicals of each component is determined respectively, and the component with the strongest scavenging capacity for the two free radicals (i.e. SDF) is screened out. Further, the free radical-enzyme synergistic extraction method is adopted, the SDF dissolution rate is taken as a response value, the optimal parameters of extraction are optimized through single factor test and Box-Behnken test design, and the wheat sprout functional dietary fiber with high yield and strong biological activity is obtained. The wheat sprout SDF extracted by the method can enhance the life and antioxidant enzyme activity of Caenorhabditis elegans under oxidative stress, increase the anti-apoptotic capacity, regulate the intestinal flora of high-fat diet induced obese C57BL / 6J mice, the filter residue (mainly IDF) after extraction of the wheat sprout SDF has good water binding capacity, water holding capacity, adsorption capacity of cholesterol and sodium cholate, can be used for functional food additives to improve food texture and nutritional characteristics, can also be used in the field of health care to assist in reducing serum cholesterol level, preventing arteriosclerosis, promoting defecation, preventing constipation and colorectal cancer, etc., and realizes high-quality reuse of the wheat sprout.

[0006] The technical scheme adopted by the application is:

[0007] In a first aspect, the application provides a wheat sprout functional dietary fiber, which comprises soluble dietary fiber (SDF) and insoluble dietary fiber (IDF), and the main components of the soluble dietary fiber include guluronic acid, mannose, ribose, galacturonic acid, galactosamine, glucose, galactose, xylose, arabinose and fucose. Among them, galacturonic acid, galactosamine, glucose and arabinose are main monosaccharides; and the wheat sprout functional dietary fiber is an oligosaccharide composed of 2-10 monosaccharides with a weight average molecular weight of 351 Da-1736 Da.

[0008] Further, the molar ratio of guluronic acid, mannose, ribose, galacturonic acid, galactosamine, glucose, galactose, xylose, arabinose and fucose in the soluble dietary fiber is 3.00:1.08:1.20:4.08:4.90:4.15:1.00:1.67:4.60:2.04.

[0009] In a second aspect, the present application provides a method for preparing functional dietary fiber from wheat sprout, which comprises:

[0010] (1) adding hydrogen peroxide and deionized water into the mixture of wheat sprout powder and cellulase, and placing the mixture into a constant temperature oscillator, adjusting the parameters of the oscillator to 40-100℃ and 200rpm, and oscillating reciprocally for 20-120min; after the extraction, placing the mixture into a boiling water bath at 100℃ for 3min to inactivate the enzyme, and centrifuging at 8000rpm for 20min to obtain the precipitate and supernatant;

[0011] (2) taking the supernatant of step (1), and concentrating the supernatant to one fifth of the original volume at 60℃ by rotary evaporation, then slowly adding the concentrated supernatant into 95% ethanol, and standing at 4℃ for 12h, and centrifuging at 3000rpm for 10min, and taking the precipitate, and dissolving the precipitate in deionized water, and removing the protein by Sevag method (chloroform: n-butanol = 4:1, V:V) until there is no absorption peak at 280nm, and freeze-drying (initial temperature -30℃, vacuum degree 80Pa) to obtain the purified wheat sprout SDF;

[0012] (3) taking the precipitate of step (1), washing, drying, crushing and sieving to obtain the wheat sprout IDF powder.

[0013] Further, the cellulase is added in step (1) in an amount of 0.1-1.8% based on the mass of the wheat sprout powder, preferably 0.4-0.8%; the hydrogen peroxide is added in the form of 30% aqueous solution, and the final concentration is 0.1-2%, preferably 0.6%; the total volume of the hydrogen peroxide and deionized water is 20-65mL / g based on the mass of the wheat sprout, preferably 40mL / g.

[0014] Further, the volume ratio of ethanol to the concentrated supernatant is 4:1 in step (2).

[0015] Further, the washing in step (3) refers to washing the precipitate with deionized water for 2-3 times to remove the residual soluble impurities, and centrifuging (8000r / min, 15min) after fully stirring each time, and discarding the supernatant; the drying refers to drying the washed wet precipitate by placing the precipitate on a petri dish and drying in an oven at 70℃ until the weight is constant; and the sieving refers to crushing the dried block by a universal crusher, and sieving through different mesh sieves (less than 60 mesh, 60-100 mesh, 100-150 mesh, 150-200 mesh, 200-300 mesh) to obtain powders with different particle sizes.

[0016] In a third aspect, the present application provides the use of functional dietary fiber from wheat sprout in preparing antioxidant products, which can effectively scavenge active oxygen and / or active nitrogen free radicals. The functional dietary fiber from wheat sprout is preferably soluble dietary fiber.

[0017] In a fourth aspect, the present application provides a use of the functional dietary fiber of wheat sprout in the preparation of an anti-aging or anti-apoptosis product, which can enhance the lifespan of Caenorhabditis elegans under oxidative stress and reduce the degree of cell apoptosis.

[0018] In a fifth aspect, the present application provides a use of the functional dietary fiber of wheat sprout in the preparation of an intestinal flora modulator, which can improve the intestinal flora imbalance of obese mice caused by high-fat feed and promote the proliferation of more beneficial bacteria.

[0019] Further, the modulator is a mixture of IDF and SDF in a mass ratio of 7:3.

[0020] In a sixth aspect, the present application also provides a use of the functional dietary fiber of wheat sprout as a food additive.

[0021] The IDF prepared by the method of the present application has good water binding capacity, water holding capacity, and ability to adsorb cholesterol and sodium cholate, and can be used in baked foods (e.g., to increase the water holding capacity of bread and biscuits, prolong the shelf life, and reduce the negative impact on the cardiovascular system by adsorbing cholesterol), low-fat foods (to simulate the taste of fat by using its high water holding capacity and swelling power, reduce the amount of oil added, and be suitable for low-fat dairy products or meat products), meal replacement products (to assist in weight management by delaying gastric emptying and increasing satiety), cholesterol reduction and cardiovascular health maintenance (the properties of adsorbing bile acids and cholesterol can help reduce serum cholesterol levels and prevent arteriosclerosis), intestinal health (to promote defecation, reduce toxin retention, prevent constipation and colorectal cancer), and the like.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] 1. In the case where the active ingredients in the tracked wheat sprout are unknown, the present application uses the water extraction and alcohol extraction processes in the solvent extraction method of classical prescriptions in the motherland, and in combination with the pharmacological test of active oxygen and active nitrogen radical scavenging capacity, to extract all the total ingredients of the wheat sprout powder with different solvents (crude extract) to the maximum extent, and on the basis of determining that the crude extract has the active oxygen and active nitrogen radical scavenging capacity, to gradually divide and track the components with the most concentrated effective ingredients, and to determine that the active ingredients in the wheat sprout in the extract are SDF. This method can avoid the loss of effective ingredients in the extraction process of the wheat sprout.

[0024] 2、The present application is based on the process of determining the active ingredient in wheat sprout extract as SDF, and using the synergistic wall-breaking-extraction method of biological enzymes and free radicals to extract wheat sprout SDF. The cellulase effectively breaks the cell wall of the wheat sprout, and hydrogen peroxide is a clean and efficient oxidizing agent and a typical environmental protection agent, and is also one of the most commonly used reagents for generating ·OH. The wheat sprout is first broken by biological enzymes, and then hydrogen peroxide is used to generate strong ·OH at a certain temperature, which can further effectively break the wall and has the advantages of unique decolorization, etc. The process is green and environmentally friendly, easy to operate, and the SDF dissolution rate can be as high as 12.90%. Compared with traditional hot water extraction of wheat sprout SDF, it is increased by 3.488 times, and compared with single cellulase extraction, it is increased by 1.589 times. The SDF extracted by the enzyme and hydrogen peroxide method has no odor and good sensory properties, and does not need to be decolorized. The low concentration of hydrogen peroxide used in the operation is easy to decompose into water and oxygen after the operation is completed, and will not leave any residues during the operation. The whole operation process does not introduce impurities, has no pollution, has low requirements for equipment and low energy consumption, and has many advantages.

[0025] 3、The wheat sprout SDF prepared by the method of the present application has the ability to scavenge active oxygen (hydroxyl radical) and active nitrogen (ABTS radical), and the effect is equivalent to that of hot water extraction and enzyme extraction. It can also enhance the lifespan of Caenorhabditis elegans under oxidative stress, reduce the degree of apoptosis of Caenorhabditis elegans under oxidative stress, improve the intestinal flora imbalance state of obese mice caused by high-fat feed, and promote the proliferation of more beneficial bacteria.

[0026] 4、After extracting the wheat sprout SDF, the IDF is compared with the traditional hot water extraction SDF, and the binding water force of the traditional hot water extraction SDF is increased by 1.078 times; the adsorption capacity of cholesterol is increased by 1.324 times. After the IDF of the wheat sprout SDF is extracted, the IDF is mixed with inulin at a ratio of 1:1, and when the powder particle size is greater than 60 meshes, the water holding capacity is increased by 1.444 times compared with the wheat sprout IDF, the water binding capacity is increased by 1.109 times compared with the wheat sprout IDF, and the oil holding capacity is equivalent to that of the wheat sprout IDF. When the powder particle size is 200-300 meshes, the cholesterol (pH 7.0) adsorption capacity is increased by 2.004 times compared with the wheat sprout IDF, and the cholesterol (pH 2.0) adsorption capacity is increased by 1.981 times compared with the wheat sprout IDF. The improvement of these functional properties can be applied in baked foods, low-fat foods, meal replacement products and other foods, and is helpful for reducing cholesterol and maintaining cardiovascular health, and is helpful for intestinal health.

[0027] 5. Wheat sprout SDF can make normal mice intestinal flora quickly ferment to produce SCFAs, and wheat sprout IDF+SDF can make obese mice intestinal flora quickly ferment to produce SCFAs. Since the intestinal flora of obese mice has been disordered, the appropriate proportion of IDF and SDF (7:3) is more conducive to intestinal flora fermentation to produce SCFAs than pure SDF, and can improve the imbalance of intestinal flora of high-fat diet induced obese mice. (IV) DESCRIPTION OF DRAWINGS

[0028] Figure 1 Figure 1 shows the extraction process of wheat sprout and the proportion of solid content.

[0029] Figure 2 Figure 2 shows the clearance rate of hydroxyl radicals (A) and IC value of clearance rate (B) of each extract of wheat sprout in Example 1. 50

[0030] Figure 3 Figure 3 shows the ABTS clearance ability (A) and IC value of clearance rate (B) of each extract of wheat sprout in Example 1. + 50

[0031] Figure 4 Figure 4 shows the effect of different factors (extraction temperature, time, hydrogen peroxide concentration, E / S, liquid-solid ratio) on the extraction rate of wheat sprout SDF.

[0032] Figure 5 Figure 5 shows the response surface diagram of wheat sprout SDF extraction.

[0033] Figure 6 Figure 6 shows the HPLC chromatogram of monosaccharide mixed standard (A) and wheat sprout SDF (B); 1, guluronic acid; 2, mannuronic acid; 3, mannose; 4, glucosamine; 5, ribose; 6, rhamnose; 7, glucuronic acid; 8, galacturonic acid; 9, galactosamine; 10, glucose; 11, galactose; 12, xylose; 13, arabinose; 14, L-fucose.

[0034] Figure 7 Figure 7 shows the Fourier infrared spectrum of wheat sprout SDF.

[0035] Figure 8 Figure 8 shows the HPGPC spectrum (A) and molecular weight distribution diagram (B) of wheat sprout SDF.

[0036] Figure 9 Figure 9 shows the surface characteristic electron microscope diagram of wheat sprout SDF; from A to C, the magnification is ×1000, ×5000 and ×10000, respectively.

[0037] Figure 10 ​​​Survival curves of the effect of wheat sprout SDF on the survival rate of nematodes under oxidative stress (A) and bar charts of the effect on average lifespan (B); the number of nematodes was 30 (n = 30), and three independent experiments were repeated.

[0038] Figure 11 Effect of wheat sprout SDF on SOD activity (A), CAT activity (B), and GSH (C) levels of C. elegans under oxidative stress.

[0039] Figure 12 Effect of different concentrations of wheat sprout SDF on apoptosis of C. elegans (the same letter indicates no difference between groups, p > 0.05; different letters indicate significant differences between groups, p < 0.05).

[0040] Figure 13 Dynamic changes in pH values of normal mouse intestinal flora fermented with wheat sprout SDF at different time points.

[0041] Figure 14 Dynamic changes in pH values of obese mouse intestinal flora fermented with wheat sprout SDF at different time points.

[0042] Figure 15 Statistical analysis of the effect of wheat sprout SDF / IDF on normal mouse intestinal flora; A represents Bray-Curtis similarity analysis, and B represents non-metric multidimensional scaling analysis.

[0043] Figure 16 Heat map (A) and relative abundance of flora composition (B) of the effect of wheat sprout SDF / IDF on normal mouse intestinal flora at the phylum level.

[0044] Figure 17 Effect of wheat sprout SDF / IDF on normal mouse intestinal flora composition at the genus level.

[0045] Figure 18 Statistical analysis of the effect of wheat sprout SDF / IDF and probiotic PB on high-fat diet-induced obese mouse intestinal flora; A represents Bray-Curtis similarity analysis, and B represents non-metric multidimensional scaling analysis.

[0046] Figure 19 Heat map (A) and relative abundance of flora composition (B) of the effect of wheat sprout SDF / IDF on obese mouse intestinal flora at the phylum level.

[0047] Figure 20 Effect of wheat sprout SDF / IDF on obese mouse intestinal flora composition at the genus level.

[0048] Figure 21Water holding capacity of wheat sprout / IDF with different particle size (a, b, c represent that the water holding capacity of wheat sprout / IDF with different particle size is significantly different, p<0.05).

[0049] Figure 22 Bound water capacity of wheat sprout / IDF with different particle size (a, b, c represent that the bound water capacity of wheat sprout / IDF with different particle size is significantly different, p<0.05).

[0050] Figure 23 Oil holding capacity of wheat sprout / IDF with different particle size (a, b, c, d represent that the oil holding capacity of wheat sprout / IDF with different particle size is significantly different, p<0.05).

[0051] Figure 24 Cholic acid sodium adsorption capacity of wheat sprout / IDF with different particle size (a, b, c, d represent that the cholic acid sodium adsorption capacity of wheat sprout / IDF with different particle size is significantly different, p<0.05).

[0052] Figure 25 Cholesterol adsorption capacity of wheat sprout / IDF with different particle size at pH 2.0 (a, b, c, d represent that the cholesterol adsorption capacity of wheat sprout / IDF with different particle size is significantly different, p<0.05).

[0053] Figure 26 Cholesterol adsorption capacity of wheat sprout / IDF with different particle size at pH 7.0 (a, b, c, d represent that the cholesterol adsorption capacity of wheat sprout / IDF with different particle size is significantly different, p<0.05).

[0054] Figure 27 Physicochemical properties of wheat sprout IDF mixed with inulin. (Five) Specific embodiments

[0055] The application will be further described in conjunction with specific embodiments, but the protection scope of the application is not limited to this:

[0056] Materials and reagents used in the embodiments of the application:

[0057] Wheat sprout powder was purchased from Taobao.

[0058] Phenol, concentrated sulfuric acid, hydrochloric acid, and hydrogen peroxide were purchased from Zhejiang Changqing Chemical Co., Ltd. The water used in the experiment was deionized water. GAM medium (without glucose and soluble starch) was purchased from Qingdao Haibo Biological Co., Ltd. Inulin was produced by Beijing Tongren Tang. Cellulase (activity 10,000 U / g) was purchased from Shanghai Maikelin Biochemical Technology Co., Ltd. Superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) kits were purchased from Nanjing Jiancheng Biological Engineering Institute.

[0059] Caenorhabditis elegans N2 wild strain and Escherichia coli OP 50 Donated by the College of Animal Science, Zhejiang University.

[0060] C57BL / 6J male mice were purchased from Shanghai Slek Experimental Animal Responsibility Co., Ltd., license number: SCXK (Shanghai) 2022-0004.

[0061] Experimental instruments used in the embodiments of the present application:

[0062] JP-250A-2 high-speed multifunctional pulverizer, Shanghai Jiupin Trade Co., Ltd.; XMTD-8222 electric heating air drying oven, Shanghai Jinghong Experimental Instrument Co., Ltd.; JA2003 electronic analytical balance, Shanghai Shunyu Hengping Scientific Instrument Co., Ltd.; L535-1 low-speed centrifuge, Hunan Xiangyi Experimental Instrument Development Co., Ltd.; TG16-WS table type high-speed centrifuge, Hunan Xiangyi Centrifuge Instrument Co., Ltd.; UV1800PC ultraviolet visible spectrophotometer, Shanghai Aozan Scientific Instrument Co., Ltd.; SHZ-D(III) circulating water type multi-purpose vacuum pump, Zhengzhou Keli Instrument and Equipment Co., Ltd.; VS-840K-U clean bench, Suzhou Antai Air Technology Co., Ltd.; Intelligent biochemical incubator, Ningbo Haishu Saifu Experimental Instrument Factory; Keyence RE-52 rotary evaporator, Shanghai Yalong Biochemical Instrument Factory; HBS-1096C enzyme label analyzer, Nanjing Detong Biological Technology Co., Ltd.

[0063] The experimental data in the embodiments of the present application are expressed as Means±SD, and the significant difference analysis is carried out by using IBM SPSS Statistics 25 software single factor variance analysis (ANOVA) in variance homogeneity test, LSD multiple comparison analysis, and p<0.05 is a significant difference.

[0064] The room temperature referred to in the embodiments of the present application refers to 25-30℃.

[0065] Example 1, dynamic tracking of active ingredients in the extraction process of wheat sprout

[0066] 1, component extraction of wheat sprout

[0067] The main solvent extraction method has the following three kinds. Lipophilic organic solvent extraction method: generally refers to the water-immiscible organic solvents, such as benzene, chloroform, ethyl acetate, etc. These solvents have strong selectivity, not easy to extract small soluble impurities, but most of them are volatile, and toxic, flammable and explosive, generally not used. Alcohol extraction method: commonly used are ethanol and methanol, since methanol is toxic, expensive, generally choose ethanol. Ethanol has good solubility, strong ability to penetrate plant cells, and the extract is not easy to mildew and easy to store, it is a commonly used solvent for extracting total ingredients. Water extraction method: traditional Chinese medicine decoction generally uses water extraction, which is one of the methods necessary for the screening of classical prescriptions. Water is the cheapest and most polar solvent, which can not only extract the hydrophilic components, but also extract some small polar substances due to the mutual solubilization of various plant components, and is one of the most commonly used solvents.

[0068] Combining the advantages and disadvantages of the three solvent extraction methods, we combine water extraction and alcohol extraction to maximize the extraction of total ingredients from wheat seedlings (initial extract), and to minimize the loss of active ingredients during extraction.

[0069] Referring to the flowchart of Figure 1 , the wheat seedling powder is extracted according to the following steps:

[0070] (1) Take 12g of purchased wheat seedling powder (passed through a 200 mesh sieve), add 360mL of deionized water at a ratio of 1g / 30mL, and extract at a temperature of 70℃ for 2h, centrifuge at 8000rpm for 20min, concentrate the supernatant to 30% of the original volume by rotary evaporation, and store at -4℃ as the water extract. The precipitate is alcohol extracted at room temperature with 95% edible alcohol at a ratio of 1g / 4mL for 24h, then centrifuged at 3000rpm for 20min, and the supernatant is removed by rotary evaporation at 55℃, then mixed with the water extract to obtain extract 1.

[0071] (2) Extract the extract 1 obtained in step (1) with petroleum ether at room temperature at a volume ratio of 1:4, separate the layers after standing, repeat the extraction 4 times, then combine the upper petroleum ether layer and the lower water layer, and remove the petroleum ether from the upper petroleum ether layer by rotary evaporation at 50℃ to obtain extract 2.

[0072] (3) The lower water layer in step (2) is extracted with chloroform at room temperature at a volume ratio of 1:4, the layers are separated after standing, the lower chloroform layer and the upper water layer are combined after repeating the extraction 4 times, and the chloroform is removed from the lower chloroform layer by rotary evaporation at 50℃ to obtain extract 3.

[0073] (4) Add 4 times the volume of ethanol to the upper water layer in step (3) for alcohol precipitation, centrifuge at 1000rpm for 5min to obtain supernatant and precipitate, dissolve the obtained precipitate in water, and rotary evaporate at 55℃ until no ethanol smell is obtained to obtain extract 4.

[0074] (5) Step (4) supernatant 55°C rotary evaporation to remove ethanol to obtain the extract 5.

[0075] 2, the solid content of the extract was detected

[0076] In order to have a general understanding of the whole process of wheat sprout powder extraction, it is necessary to measure the solid content of each component in each step of the extraction process, which accounts for the ratio of raw material wheat sprout powder (yield): weigh the empty glass weighing bottle weight as M1; respectively, 5 mL of water extract prepared in step 1, extract 1 ~ extract 5 into glass weighing bottle, 50°C oven drying, weighing each group of glass weighing bottle weight as M2. The solid mass in each extract is (M2-M1); the solid concentration is C (g / L) = (M2-M1) / 0.005. The results are shown in Figure 1 .

[0077] From Figure 1 It can be seen that after water extraction and alcohol extraction of wheat sprout powder, nearly 27% of the soluble ingredients of the raw material can be obtained, that is, extract 1. After the extract 1 is sequentially fractionated by different polar organic solvents, extract 2, 3, 4 and 5 are obtained respectively. Among them, extract 5 accounts for the highest, reaching 16.3% of the initial raw material weight; followed by extract 4, accounting for 6.96% of the initial raw material weight; the content of extract 2 and 3 is low. It can be seen that the highest proportion in wheat sprout is insoluble component (mainly insoluble dietary dietary, accounting for 63%), which is polar material, weak polar and non-polar component with low content. The following further studies the ability of removing active oxygen (hydroxyl radical) and active nitrogen (ABTS + ) of each component.

[0078] 3, the detection of each extract removing active oxygen (hydroxyl) radical

[0079] Sample solution: according to the solid concentration of each extract in step 2 detection, the solid concentration is continuously diluted by equal multiples to 6 concentrations (0.01625, 0.07688, 0.4, 1.23, 3.3 g / L), namely sample solution.

[0080] Sample group: 1 mL of sample solution with different concentrations above, 2 mL of 1.8 mM ferrous sulfate aqueous solution, 1.5 mL of 1.8 mM salicylic acid aqueous solution, 0.1 mL of 0.03% hydrogen peroxide were added into test tube. Control group: replace hydrogen peroxide in sample group with equal volume of deionized water. Blank group: replace sample solution in sample group with equal volume of deionized water.

[0081] Each group was mixed uniformly, and the absorbance was measured at 510 nm after cooling at 37°C water bath for 30 min.

[0082] Hydroxyl radical scavenging rate (%) = [1-(A 样 -A 对照 ) / A 空白 ] x 100%

[0083] Wherein, A 样 represents the absorbance of the sample, A 对照 refers to the system without adding hydrogen peroxide, A 空白 refers to the system without adding the sample.

[0084] The hydroxyl radical scavenging rate curves of extract 1-5 are shown in Figure 2 A, the IC 50 values of extract 1, 4 and 5 are shown in Figure 2 B.

[0085] As can be seen from Figure 2 , the scavenging ability of the crude extract (i.e. extract 1) on hydroxyl radicals increases continuously with the increase of the concentration, which is concentration-dependent. When the concentration of extract 1 is 1.8 mg / mL, the scavenging rate on hydroxyl radicals reaches 31.21%, indicating that the crude extract has a certain ability to scavenge hydroxyl radicals. After the extract 1 is fractionated into four components according to the polarity, the scavenging ability of extract 2 on hydroxyl radicals is basically unchanged with the increase of the concentration, and the scavenging rate is all below 21%; the scavenging ability of extract 3 on hydroxyl radicals shows a fluctuating trend with the increase of the concentration, and the scavenging rate is all below 30%. Since the scavenging ability of extract 2 and extract 3 on hydroxyl radicals has no concentration dependence and the scavenging rate is all below 30%, the IC 50 cannot be calculated. The scavenging ability of extract 4 and extract 5 on hydroxyl radicals increases continuously with the increase of the concentration, which is concentration-dependent. The IC 50 of extract 1 and extract 5 for scavenging hydroxyl radicals is 3.211 mg / mL and 11.82 mg / mL respectively, i.e. the concentrations required to reach 50% of the scavenging rate of hydroxyl radicals are 3.211 mg / mL and 11.82 mg / mL respectively. The IC 50 of extract 4 is only 1.292 mg / mL, indicating that among the four components of the crude extract, the scavenging ability of extract 4 on hydroxyl radicals is the strongest.

[0086] 4. Detection of the scavenging of active nitrogen (ABTS) radicals by each extract

[0087] Sample solution: according to the original concentration of the solid of each extract detected in step 2, the solid concentration is continuously diluted by equal multiples with deionized water into 6 concentrations (0.01625, 0.04, 0.0825, 0.2, 0.615, 3.2 g / L), i.e. the sample solution.

[0088] Potassium persulfate solution: 2.6 mmol / L potassium persulfate solution is prepared with deionized water.

[0089] ABTS working solution: Prepare a 7.4 mmol / L ABTS solution with deionized water, mix it with pH 7.4 phosphate buffer at a volume ratio of 1:1, and store it at room temperature in the dark for 12 hours to obtain the ABTS working solution.

[0090] Add reagents according to the following groupings and mix thoroughly. Let stand for 6 minutes in the dark, then measure the absorbance at 734 nm. Details are as follows:

[0091] Sample group: 0.2 mL sample solution + 0.8 mL ABTS working solution, absorbance value recorded as A1;

[0092] Blank group: 0.2 mL deionized water + 0.8 mL ABTS working solution, absorbance value recorded as A2.

[0093] The ABTS free radical scavenging rate is calculated using the formula: Scavenging rate (%) = [(A2-A1) / A2] × 100%

[0094] Extract 1 and extracts 2, 3, 4, and 5 for ABTS + The clearance rate and the IC of clearance rate 50 Worth seeing Figure 3 A and B.

[0095] Depend on Figure 3 It can be seen that the crude extract (i.e., extract 1) has a significant effect on ABTS. + The scavenging ability of the crude extract increases continuously with increasing concentration, exhibiting a concentration-dependent effect. When the concentration of extract 1 is 0.66 mg / mL, the scavenging rate of hydroxyl radicals reaches 53.47%, indicating that the crude extract has a strong scavenging ability for ABTS. + Ability. After extract 1 was fractionated into four components (2, 3, 4, and 5) according to polarity, extract 2 of different concentrations basically showed good effect on ABTS. + None of them had the ability to remove ABTS; the extract 3 was effective against ABTS. + The scavenging ability increased slowly with increasing concentration, and the scavenging rate was below 10% for both extracts. IC50 values ​​could not be calculated for extracts 2 and 3. 50 Extracts 4 and 5 were compared with ABTS. + The scavenging ability of these compounds increases continuously with increasing concentration, exhibiting a concentration-dependent effect. Extracts 1, 4, and 5 were used to scavenge ABTS. + IC 50 The values ​​were 0.5959 mg / mL, 2.396 mg / mL, and 0.2665 mg / mL, respectively. The IC50 values ​​for extracts 1 and 5 were... 50 Similarly, the IC50 of extract 4 50 Slightly higher, but still has a strong ability to clear ABTS. + ability.

[0096] In combination with the above active oxygen and active nitrogen radical scavenging ability, the extraction solutions 4 and 5 show a dose-effect relationship in the scavenging effect on the two kinds of radicals in the concentration range, among which the extraction solution 5 has the strongest scavenging effect on the active nitrogen radical, and the extraction solution 4 has the strongest scavenging effect on the active oxygen radical. According to the classification principle of the extraction solution 1, the extraction solution 5 is an alcohol-soluble component, and the extraction solution 4 is a water-soluble macromolecule. Considering the use cost, safety and other factors in actual application, the following focuses on the research of the extraction solution 4. In combination with the classification process and the component composition of the wheat germ seedlings, it is speculated that the macromolecular substance with strong radical scavenging ability in the extraction solution 4 should be soluble dietary fiber (SDF). The extraction process of the wheat germ seedling SDF will be further optimized, the possible molecular structure information will be studied, and the function will be evaluated.

[0097] Example 2, Optimization of Wheat Germ Seedling SDF Extraction Process

[0098] According to the speculation in Example 1 that the component with strong active oxygen and active nitrogen scavenging ability in the wheat germ seedlings is SDF, the extraction process parameters of the wheat germ seedling SDF are optimized by using the free radical-enzyme synergistic method.

[0099] 1. Single factor test of wheat germ seedling SDF dissolution

[0100] (1) Temperature

[0101] 2g of wheat germ seedling powder was weighed, 0.02g of cellulase was added according to the addition amount of E / S (cellulase accounting for the mass percentage of wheat germ seedling powder) of 1.0%, 4mL of hydrogen peroxide (30%) and 96mL of deionized water were added, so that the concentration of hydrogen peroxide was 1.2%, the liquid to material ratio was 50:1 (mL / g), and the temperature was controlled at 40℃, 50℃, 60℃, 70℃, 80℃, 90℃ or 100℃, respectively, and the extraction time was controlled for 60min. After the extraction was completed, the enzyme was inactivated in a 100℃ boiling water bath for 3min. Centrifugation was performed at 8000rpm for 20min, 1.0mL of supernatant was taken, and the absorbance value of the supernatant at 480nm was detected according to GB / T 15672-2009. The SDF concentration in the supernatant was calculated according to the standard curve of glucose concentration and absorbance value, the standard equation was y=9.2133x+0.1298, R 2 =0.9986, y represented the absorbance value, x represented the SDF concentration, and was converted to the yield of wheat germ seedling SDF according to formula (1).

[0102] Yield of wheat germ seedling SDF (%)=(C×V×f) / m×100% formula (1)

[0103] In formula (1), C is the concentration of wheat sprout SDF (mg / mL) converted by standard equation; V is the volume of supernatant (mL); f is the dilution multiple; and m is the mass of wheat sprout powder sample (mg).

[0104] (2) Hydrogen peroxide concentration

[0105] In step (1), the temperature was fixed at 60°C, and the hydrogen peroxide concentration was changed to 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8% and 2.0% respectively, and other operations were the same.

[0106] (3) Liquid-to-material ratio

[0107] In step (1), the temperature was fixed at 60°C, and the liquid-to-material ratio was changed to 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1 mL / g respectively, and other operations were the same.

[0108] (4) Enzyme addition amount

[0109] In step (1), the temperature was fixed at 60°C, and the enzyme addition amount was changed to 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.2%, 1.4%, 1.6% or 1.8% respectively, and other operations were the same.

[0110] (5) Extraction time

[0111] In step (1), the temperature was fixed at 60°C, and the extraction time was controlled to be 20, 40, 60, 80, 100 or 120 min respectively, and other operations were the same.

[0112] The effects of extraction temperature, time, hydrogen peroxide concentration, enzyme addition amount and liquid-to-material ratio on the extraction yield of wheat sprout SDF are shown in Table Figure 4 .

[0113] As can be seen from Table Figure 4 , with the increase of extraction temperature from 40°C to 100°C, the extraction yield of wheat sprout SDF showed a fluctuating trend of first decreasing, then increasing and then decreasing, but there was no significant difference between each level. It was speculated that the best action temperature of cellulase and the effect of temperature increase on accelerating the dissolution of SDF counteracted each other, that is, the effect of temperature increase and the effect of enzyme were different. This factor did not have a vertex on the response value, so it was not optimized, and therefore the extraction temperature was determined to be 40°C in the subsequent experiment.

[0114] The yield of SDF from wheat seedlings increased with the increase of extraction time from 20 min to 60 min. When the extraction time increased to 100 min, the yield of SDF from wheat seedlings decreased. When the extraction time increased to 100 min, the yield of SDF from wheat seedlings increased again, but there was no significant difference compared with 40 min, 60 min and 80 min. Therefore, 60 min was used as the center point in the subsequent optimization experiment.

[0115] With the increase of hydrogen peroxide concentration from 0.1% to 1.8%, the yield of SDF from wheat seedlings showed a fluctuating trend of first increasing, then decreasing, then increasing and then decreasing. Except for the hydrogen peroxide concentration of 1.4%, there was no significant difference between the other levels, and there was also no significant difference between the yield of SDF from wheat seedlings with hydrogen peroxide concentration of 0.6% and 1.4%. Therefore, this factor does not need to be optimized, and the hydrogen peroxide concentration is determined as 0.6% in the subsequent experiment.

[0116] The yield of SDF from wheat seedlings showed a fluctuating trend with the increase of enzyme addition amount from 0.1% to 1.8%. When the enzyme addition amount was 0.8%, the yield of SDF from wheat seedlings was the highest. Therefore, 0.8% was used as the center point in the subsequent optimization experiment.

[0117] When the liquid-solid ratio increased from 20 mL / g to 40 mL / g, the yield of SDF from wheat seedlings increased significantly. When the liquid-solid ratio of the system exceeded 40 mL / g, the yield of SDF from wheat seedlings showed a fluctuating trend. 40 mL / g was selected as the center point in the subsequent optimization experiment.

[0118] 2. Response surface analysis experiment

[0119] On the basis of the above single factor experiment, the three factors that most significantly affected the yield of SDF from wheat seedlings (extraction time, enzyme addition amount, and liquid-solid ratio) were selected as independent variables, and the yield of SDF from wheat seedlings was used as the response value. Using Design-Expert.V8.0.6 software, Box-Behnken Design (three factors and three levels) was used to design the response surface analysis experiment according to the principle of combination experiment design. The factor levels of the response surface experiment are shown in Table 1, and the experimental design and results are shown in Table 2.

[0120] Table 1 Factor level table for optimization of composite enzyme method for extracting SDF from wheat seedlings

[0121] Coding level A: extraction time (min) B: enzyme addition amount (%) C: liquid material ratio (mL / g) -1 20 0.4 30 0 60 0.8 40 1 100 1.2 50

[0122] Table 2 Experimental design and results of SDF extraction from wheat seedlings

[0123]

[0124] The multiple regression fitting was made to the test data in Table 2, and the regression equation with the yield of wheat sprout SDF (Y) as the response value was obtained:

[0125] Y = 12.09 + 0.794A + 0.439B + 0.074C - 0.038AB - 0.108AC + 0.522BC - 0.879A 2 -1.01B 2 -2.06C 2

[0126] The variance analysis was made to the regression equation, and the results are shown in Table 3.

[0127] Table 3 Regression model analysis of the extraction parameters of wheat sprout SDF

[0128]

[0129]

[0130] The analysis of Table 3 shows that the p value is used as a tool to check the significance of each coefficient, the model p value is <0.0001, indicating that the model is significant (p<0.01). R 2 is 0.9906, indicating that the predicted value is well fitted with the simulation test value within the range of the test parameters; the adjusted R 2 is 0.9786, indicating that the equation can explain 97.86% of the response value change, and the fitting degree is good. The p value of the misfit term is 0.1816, which is not significant, indicating that the residual error is caused by random error, which is not significant (p>0.05) relative to the pure error; the C.V value is 2.14%, indicating that the model has high reliability. Therefore, the regression equation can be used to predict the test results. Among the three factors, the linear terms A, B and the quadratic terms A 2 , B 2 , C 2 have significant effects on the response value curve; the interaction terms AB and AC have no significant effects on the response value surface, and the interaction term BC has a significant effect on the response value surface, indicating that there is a synergistic effect between the enzyme addition amount and the liquid-solid ratio. The influence order of the three factors on the yield of wheat sprout SDF is A>B>C.

[0131] The response surface and contour graph of the interaction factors according to the regression equation are shown in Figure 5 .

[0132] The large slope of the response surface graph indicates that the factor has a large effect on the response value, and the dense contour is elliptical, indicating that the interaction of the two factors is large, while the gentle slope and the circular contour are opposite. Figure 5 Among them, the interaction contour of BC tends to be elliptical, indicating that there is an interaction between the two.

[0133] The optimal conditions for model optimization are: extraction time of 69.463 min, enzyme addition amount of 0.829%, and liquid-to-material ratio of 40.146 mL / g, and under the conditions, the wheat sprout SDF yield can reach 12.255%. In combination with the actual laboratory conditions, the optimal adjustment is: extraction time of 70 min, enzyme addition amount of 0.8%, and liquid-to-material ratio of 40 mL / g. In order to further verify the effectiveness and accuracy of the model and the actual situation, three parallel experiments were carried out under the extraction conditions optimized by the response surface experiment, and the wheat sprout SDF yield can reach 12.90% ± 0.19%, which is consistent with the predicted value, indicating that the response surface analysis method is reliable and well fitted with the actual situation, thereby verifying the effectiveness of the regression equation.

[0134] Example 3, extraction and separation and purification of wheat sprout SDF and IDF

[0135] (1) Under the optimal process parameters optimized in Example 2, the wheat sprout SDF was extracted: 2 g of wheat sprout powder was weighed, 0.016 g of cellulase was added at an E / S (cellulase accounting for the mass percentage of wheat sprout powder) of 0.8%, 1.6 mL of hydrogen peroxide (30%) and 78.4 mL of deionized water were added to make the hydrogen peroxide concentration 0.6%, the liquid-to-material ratio was 40:1 (mL / g), and the mixture was placed in a constant temperature oscillator, the oscillator parameters were adjusted to 40°C and 200 rpm, and the reciprocating rotary oscillation extraction was carried out for 70 min. After the extraction was completed, the enzyme was inactivated in a 100°C boiling water bath for 3 min. Centrifugation was carried out at 8000 rpm for 20 min to obtain the precipitate and supernatant.

[0136] (2) The supernatant of step (1) was taken, concentrated to one-fifth of the original volume by rotary evaporation at 55°C, then slowly added to 95% ethanol at a ratio of 1:4 (V / V), and placed at 4°C for 12 h of alcohol precipitation, centrifuged at 3000 rpm for 10 min, and the precipitate was taken, dissolved in deionized water, and then deproteinized by the sevag method (chloroform:n-butanol=4:1, V:V) until there was no absorption peak at 280 nm, and then freeze-dried (initial temperature -30°C, vacuum degree 80 Pa) to obtain 0.15 g of purified wheat sprout SDF.

[0137] (3) The precipitate of step (1) was washed with water (the precipitate was washed with deionized water for 2-3 times to remove residual soluble impurities. Each time, the mixture was stirred thoroughly and then centrifuged at 8000 r / min for 15 minutes, and the supernatant was discarded), dried (the washed wet precipitate was spread on a petri dish and placed in a 70°C oven for drying until the weight was constant), pulverized (the dried block was pulverized with a universal pulverizer), and sieved (less than 60 mesh, 60 mesh-100 mesh, 100 mesh-150 mesh, 150 mesh-200 mesh, 200 mesh-300 mesh) to obtain a total of 0.92 g of wheat sprout IDF powder.

[0138] Example 4, Chemical composition analysis of wheat sprout SDF

[0139] The wheat sprout SDF prepared by the method of Example 3 was analyzed as follows:

[0140] 1. Monosaccharide composition of wheat sprout SDF by high performance liquid chromatography (HPLC)

[0141] Monosaccharide composition is one of the key factors affecting the physicochemical and biological activities of SDF.

[0142] Monosaccharide mixture standard: standard samples of guluronic acid, mannuronic acid, mannose, glucosamine, ribose, rhamnose, glucuronic acid, galacturonic acid, galactosamine, glucose, galactose, xylose, arabinose, and L-fucose were mixed in a molar ratio of 1:1, dissolved in 10 mL of deionized water to obtain a mixed monosaccharide standard solution of 0.4 mg / mL. When used, it was mixed with 0.6 mol / L NaOH at a ratio of 1:1 before derivatization.

[0143] Wheat sprout SDF hydrolysate: 20 mg of wheat sprout SDF sample was taken in a 10 mL hydrolysis tube, 5 mL of 2 mol / L trifluoroacetic acid (TFA) aqueous solution was added, the tube was sealed with N2 (10 L / min, 1 min), and hydrolysis was carried out in a 110°C oven for 2 h. After cooling, the cap was opened, 1 mL was taken and added with 1 mL of methanol, and then blown dry with N2 in a 70°C water bath. This was repeated twice to remove TFA. 1 mL of 0.3 mol / L NaOH solution was added to dissolve the residue, and 1 mL of wheat sprout SDF hydrolysate was obtained.

[0144] Take 400 μL of the mixed monosaccharide standard solution or wheat germ SDF hydrolysate into a 5 mL stoppered test tube, add 400 μL of 0.5 mol / L PMP (1-phenyl-3-methyl-5-pyrazolone) methanol solution (i.e., dissolve 0.4355 g of PMP in methanol and bring the volume to 5 mL), vortex to mix, and react in a 70 °C water bath for 2 h; remove and allow to cool to room temperature; add 400 μL of 0.3 mol / L HCl to neutralize (pH 6–7); add 1200 μL of water, then add an equal volume of chloroform, vortex to mix, shake, let stand, discard the chloroform phase, and repeat the extraction twice. Filter the aqueous phase through a 0.45 μm microporous membrane (aqueous system) for HPLC analysis. An Agilent 1100 high-performance liquid chromatograph with a C18 column (5 μm, 4.6 × 250 mm) was used for detection at 250 nm UV. Mobile phase A was 100 mM sodium phosphate buffer (pH = 6.7); mobile phase B was acetonitrile. The column temperature was 30 °C, the flow rate was 1 mL / min, and the injection volume was 5 μL. Gradient elution was used. At 0 min, mobile phase A accounted for 86% and mobile phase B accounted for 14%; at 9 min, mobile phase A accounted for 83% and mobile phase B accounted for 17%; at 28 min, mobile phase A accounted for 78% and mobile phase B accounted for 22%; at 29 min, mobile phase A accounted for 50% and mobile phase B accounted for 50%; and at 32 min, mobile phase A accounted for 86% and mobile phase B accounted for 14%.

[0145] The elution times of various monosaccharides in the mixed standard are as follows: Figure 6 As shown in Figure A, wheat sprout SDF samples are shown in Figure B. Through comparison... Figure 6 The elution times of monosaccharides in the mixed standard A and the elution times of wheat sprout SDF in B indicate that wheat sprout SDF is a heteropolysaccharide mainly composed of 10 monosaccharides: guluronic acid, mannose, ribose, galacturonic acid, galactosamine, glucose, galactose, xylose, arabinose, and fucose. The molar ratio of these 10 monosaccharides is 3.00:1.08:1.20:4.08:4.90:4.15:1.00:1.67:4.60:2.04, with galacturonic acid, galactosamine, glucose, and arabinose being the main monosaccharides. This shows that wheat sprout SDF contains not only neutral sugars but also a relatively high proportion of negatively charged monosaccharides (such as galacturonic acid) and positively charged monosaccharides (such as galactosamine), which is extremely rare in natural polysaccharides, suggesting that this monosaccharide composition may have certain special functions. In addition to the 14 common monosaccharides mentioned above, Figure 6 The SDF chromatogram of wheat sprouts in sample B showed a large peak between 28 and 29 min, with a content of 54.788 μmol / g. Based on the elution chromatographic conditions (non-polar C18 column as stationary phase and polar acetonitrile as mobile phase), it is speculated that the substance is tagatose.

[0146] 2. Fourier transform infrared spectroscopy analysis of SDF in wheat sprouts

[0147] Fourier transform infrared spectroscopy (FT-IR) can be used to analyze the functional groups of SDF. Weigh 2 mg of wheat sprout SDF sample and grind it thoroughly under an infrared lamp. Add dry KBr powder and continue grinding until homogeneous. After uniformly compressing, infer the mixture using an infrared spectrometer at 4000-400 cm⁻¹. -1 Scanning spectrum.

[0148] like Figure 7 It can be seen that wheat sprout SDF has seven vibrational absorption peaks at 3755.46, 3421.14, 2931.33, 1636.56, 1404.12, 1250.02 and 1045.20 cm⁻¹. SDF molecules have many hydrogen bonds and are polyhydroxy compounds. Wheat sprout SDF shows a peak at 3755 cm⁻¹. -1 With 3421cm -1 The peak belongs to the stretching vibration absorption peaks of OH and NH, originating from the hydrogen bonds between and within SDF molecules. It is an absorption peak formed by the overlap of multiple stretching vibration absorption peaks. 2931cm -1 The narrow peak nearby is due to the CH asymmetric stretching vibration of the methyl group, and the absorption peak in this region is a characteristic peak of SDF. At 1636 cm⁻¹ -1 Left and right sides and 1404cm -1 The absorption peaks around 1240 cm⁻¹ indicate the presence of NH stretching vibrations of amino groups and uronic acid in the molecule, which is consistent with the results of the monosaccharide composition analysis. -1 The peaks are CO stretching vibration peaks, with the stretching vibration of the pyran ring CO corresponding to an absorption peak at 1100-1010 cm⁻¹. The SDF of wheat sprouts is at 1045 cm⁻¹. -1 The strong absorption peak at that point can be attributed to the stretching vibration of the pyran ring of the glycosyl residue.

[0149] 3. Determination of molecular weight by gel permeation chromatography (GPC)

[0150] 5 mg of wheat germ SDF sample was dissolved in 10 mL of 50 mM sodium sulfate aqueous solution and filtered through a 0.45 μm membrane. An Agilent 1260 high-performance liquid chromatograph was used, with three columns in series: Ultrahydrogel™ 120-™ 250-™ 500 water-soluble gel columns (7.8 × 300 mm). The mobile phase was 0.1 M NaNO3 aqueous solution, and the detector was G1362A, detected at 40 °C. Twelve polyethylene glycol standards were included, with molecular weights of 326,000, 152,000, 78,300, 44,000, 25,300, 20,600, 12,600, 6,690, 4,290, 1,400, 1030, and 430 Da.

[0151] The HPGPC chart of wheat sprout SDF is shown in Figure 8. The wheat sprout SDF is mainly composed of five components with different molecular weights. According to the standard equation LogMw = -0.2779R T +10.072(R 2 = 0.9823), the weight average molecular weight Mw of the five components in the wheat sprout SDF is respectively 1736, 1012, 639, 476 and 351 Da, the number average molecular weight Mn is respectively 1701, 993, 634, 472 and 348 Da. Mw / Mn is respectively 1.020, 1.019, 1.008, 1.009 and 1.009. The closer Mw / Mn is to 1, the more concentrated the SDF molecular distribution is. At the same time, the weight average molecular weight of the five components in the SDF can be deduced that the five SDFs are composed of 2-10 monosaccharides. Studies have shown that high molecular weight leads to high molecular natural SDF difficult to cross the membrane to play its biological role, including physical properties such as solution viscosity, and more complex properties such as biological activity; low molecular weight SDF has high antioxidant activity. The free radical-enzyme mediated method is used to extract SDF in the application, and the disaccharides and oligosaccharides composed of 2-10 monosaccharides are obtained, which can expose the active site and obtain high biological activity of wheat sprout SDF.

[0152] 4. Scanning electron microscope (SEM) observation of the microstructure of wheat sprout SDF

[0153] Scanning electron microscope (SEM) is one of the most effective methods for determining the apparent physical structure of natural SDF. The method is simple, easy to operate and has little pollution to the SDF sample. The apparent structure of SDF can be directly observed to be linear or flaky, and is widely used for SDF apparent structure analysis. The morphology of the wheat sprout SDF prepared in Example 3 above was analyzed by scanning electron microscope. 0.5 mg of sample was coated with MC1000 ion sputter, and observed by German ZEISS Gemini SEM300 scanning electron microscope (SEM).

[0154] Figure 9SEM images of wheat sprout SDF amplified by different fold. The microstructure of wheat sprout SDF is smooth and dense irregular spherical structure with regular geometric shape, smooth surface and regular arrangement, and no curling phenomenon, which indicates that the morphology of wheat sprout SDF is uniform, high crystallinity or uniform molecular weight. The main intermolecular forces of macromolecular SDF include hydrogen bond, hydrophobic force and other non-covalent force. When hydrogen bond and other forces are destroyed, the conformation of SDF will change, thereby affecting its biological function. In addition, the microstructure of wheat sprout SDF has some pores and cracks, which may be due to the voids left by ice crystal sublimation during sample freeze-drying. This porous structure makes the wheat sprout SDF particles have a larger specific surface area, which can combine with more water, thereby making it have a larger polarity, good solubility and strong water molecule binding capacity.

[0155] Example 5, Effect of wheat sprout SDF on the resistance of Caenorhabditis elegans under oxidative stress

[0156] 1. Effect on the survival rate of Caenorhabditis elegans

[0157] Hydrogen peroxide is widely used as an effective oxidant for inducing oxidative stress in vivo and in vitro due to its easy metabolism into highly active hydroxyl radicals that attack organic molecules.

[0158] Preparation of 1000 mL of Caenorhabditis elegans growth medium (NGM medium): 2.5 g of proteose peptone, 3 g of NaCl, 17 g of agar, 25 mL of PBS buffer (pH 6.0, 1M), 975 mL of deionized water, after sterilization, add 1 mL of membrane filtered (0.22 μm) cholesteryl water solution (5 mg / mL), 1 mL of MgSO4 aqueous solution (1M), and 1 mL of CaCl2 aqueous solution (1M).

[0159] 1000 mL of LB liquid medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, adjusted to neutral with 5M NaOH, and distilled water as solvent.

[0160] 1000 mL of M9 buffer: 15.12 g of Na2HPO4·12H2O, 3 g of KH2PO4, 5 g of NaCl, 0.25 g of MgSO4·7H2O, and deionized water as solvent.

[0161] Nematode culture: E. coli OP50 was inoculated in LB liquid medium and cultured overnight at 37°C on a shaker to obtain E. coli OP50 bacterial solution, which was stored at 4°C for later use. Caenorhabditis elegans wild type N2 strain was inoculated on NGM medium coated with E. coli OP50 and the bacteria grew well, and incubated at 20°C for 72 h. A large number of adult and juvenile worms were observed on the medium.

[0162] C. elegans survival rate experiment: The experiment included a negative control group (M9 buffer), a positive control group (Vc, added to a final concentration of 0.5 mg / mL), and wheat sprout SDF sample groups prepared according to the method of Example 3 (added to final concentrations of 0.25, 0.5, 1, 2, and 2.5 mg / mL), with 3 replicates for each group. 1 mL of M9 buffer containing different final concentrations of samples (Vc, wheat sprout SDF) was spread onto each NGM plate. 150 μL of E. coli OP50 bacterial suspension (concentration at 10) was then added to each plate. 9 (CFU / mL). L4 stage nematodes were picked and placed on plates of each group, with 3 replicates per group, and cultured at 20℃ for 48 h. Then, the nematodes from each group were transferred to 96-well plates, and 150 μL of M9 buffer containing 2 mM hydrogen peroxide was added to each well. The plates were then cultured at 20℃, and the number of dead C. elegans was recorded every 40 min. The survival rate of C. elegans at different time points was calculated.

[0163] Depend on Figure 10 It can be seen that under hydrogen peroxide stress, the average survival time (T) of nematodes in the negative control group was... aver The duration was only 311.94 minutes, and the positive control group nematode T aver The survival time reached 438.03 min. Except for the low concentration of 0.25 mg / mL, other concentrations (0.5, 1.0, 2.0, and 2.5 mg / mL) of wheat sprout SDF all shifted the nematode survival time curve to the right and significantly prolonged the nematode T... aver (p<0.05). Compared with the negative control group, high concentrations (1.0-2.5 mg / mL) of wheat sprout SDF significantly prolonged nematode T... aver (p<0.05), and the effect was significantly better than the positive control (p<0.05); the effect of low concentration (0.25mg / mL) wheat sprout SDF was weak (p>0.05).

[0164] 2. Effects on SOD / CAT activity and GSH levels in C. elegans under oxidative stress

[0165] Methyl viologen can induce the production of reactive oxygen species (ROS) in various experimental systems, including isolated mitochondria, *C. elegans*, and mice. Therefore, methyl viologen was selected to induce ROS production in *C. elegans*, thereby inducing oxidative stress.

[0166] According to the kit instructions provided by Nanjing Jiancheng Research Institute, the activities of superoxide dismutase (SOD) and catalase (CAT) in C. elegans cultured at 20℃ for 48h in each group in step 1, as well as the level of glutathione (GSH) in C. elegans, were detected.

[0167] SOD vitality is likeFigure 11 As shown in Fig. 4A, compared with the negative control group (M9 buffer), all concentrations (0.25-2.5 mg / mL) of wheat sprout SDF could significantly enhance the SOD activity of C. elegans under stress induced by methyl viologen. With the increase of the concentration of wheat sprout SDF, the SOD activity of C. elegans showed a fluctuation phenomenon of significant decrease, significant increase and significant decrease again. The effect was optimal when the concentration of wheat sprout SDF was 2.0 mg / mL, and there was no significant difference (p>0.05) compared with the positive substance (Vc). The results showed that the specific concentration of wheat sprout SDF could remove superoxide free radicals by increasing SOD activity, and enhance the antioxidant stress ability.

[0168] CAT activity is shown in Fig. 4B. Compared with the negative control group, low concentration (0.25-1.0 mg / mL) of wheat sprout SDF could enhance the CAT activity, but there was no significant difference (p>0.05); the CAT activity of C. elegans treated with high concentration (2.0 mg / mL and 2.5 mg / mL) of wheat sprout SDF was significantly higher than that of the negative control group (p<0.05). Similar to the effect on SOD, the CAT activity of C. elegans was the strongest when the concentration of wheat sprout SDF was 2.0 mg / mL, and it was significantly better than that of the positive substance (p<0.05). The results showed that the specific concentration of wheat sprout SDF could significantly improve the decomposition of hydrogen peroxide by CAT, and enhance the antioxidant defense ability of the body. Figure 11 GSH level is shown in Fig. 4C. Compared with the negative control group, low concentration (0.25 mg / mL) and high concentration (2.5 mg / mL) of wheat sprout SDF could increase the GSH level of C. elegans, but there was no significant difference (p>0.05). The medium concentration (0.5-2.0 mg / mL) of wheat sprout SDF could significantly increase the GSH level of C. elegans (p<0.05), which was increased by 46.21%, 66.78% and 55.85% compared with the negative control group, respectively. Among them, there was no difference (p>0.05) between the effect of wheat sprout SDF at a concentration of 1.0 mg / mL and the positive control group.

[0169] Figure 11 3. Protection of C. elegans apoptosis under oxidative stress

[0170] Acridine orange (AO) fluorescent dye can bind to DNA in cells.

[0171] Acridine orange (AO) fluorescent dye can bind to DNA in cells.

[0172] ​C. elegans apoptosis assay: Nematodes grouped and cultured for 48 h according to the C. elegans survival assay method were washed with M9 buffer and transferred to Eppendorf tubes. After oxidative stress of 2 mM hydrogen peroxide (150 μL) for 2 h, the nematodes were transferred to new 1.5 mL Eppendorf tubes, and 10 μL of acridine orange working solution (prepared with M9 buffer to a 0.1 mg / mL acridine orange solution of 400 μL, followed by 100 μL containing a small amount of E. coli OP) was added (the amount does not affect the experimental results). 50 The acridine orange concentration was adjusted to 80 μg / mL using M9 buffer, and staining was performed in the dark for 1.0 h. After washing with M9 buffer, the nematodes were inoculated back onto NGM medium and incubated at 20°C for 30 min. The stained nematodes were then pipetted onto 3 cm NGM medium coated with E. coli OP50 and allowed to grow well, and incubated for 60 min (this allowed the nematodes to excrete the dye from their intestines). The nematodes were fixed with 4% paraformaldehyde for 30 min. Under a dissecting microscope, nematodes were picked up and placed on a slide, then dropped into M9 buffer for observation and image acquisition using a laser confocal fluorescence microscope. The excitation wavelength of the fluorescence microscope was 488 nm, and the blocking wavelength was 515 nm. Apoptotic cells appeared bright yellow or orange-yellow, while non-apoptotic cells appeared light green.

[0173] like Figure 12 As shown, the fluorescence intensity of the low-concentration (0.25 mg / mL and 0.5 mg / mL) wheat sprout SDF treatment groups was not significantly different from that of the negative control group (p>0.05); the fluorescence intensity of the high-concentration (1.0 mg / mL and 2.0 mg / mL) wheat sprout SDF treatment groups was significantly higher than that of the negative control group (p<0.05), indicating that the ability of wheat sprout SDF to reduce C. elegans cell apoptosis is concentration-dependent. Furthermore, the fluorescence intensity of the high-concentration (1.0 mg / mL and 2.0 mg / mL) wheat sprout SDF treatment groups was not significantly different from that of the positive control group (p>0.05), indicating that high-concentration wheat sprout SDF can effectively slow down the degree of C. elegans cell apoptosis under oxidative stress.

[0174] Example 6: Effects of wheat sprout SDF on mouse gut microbiota

[0175] 1. Prepare reagents

[0176] GAM basal medium: Weigh 49.0g of GAM medium, heat and dissolve it in 1000mL of deionized water, autoclave at 121℃ for 15min, and when cooled to about 50℃, add 1mL of sterile 0.1% vitamin K1 solution and 1mL of heme chloride solution (5mg / mL) to every 1000mL of medium, mix well and set aside.

[0177] 0.1% Vitamin K1 solution preparation: Weigh 0.01 g of Vitamin K1, put it in a conical flask, add 1 mL of Tween 80, shake the conical flask gently, then stir with a glass rod to make Vitamin K1 and Tween 80 fully mixed. After adding a small amount of deionized water, stir and shake to constant volume to 100 mL, and 0.1% Vitamin K1 solution is obtained (2-8℃ avoid light can be stored for 1 year).

[0178] 5mg / mL hematin chloride solution preparation: Weigh 0.5 g of hematin chloride and add it to the appropriate amount of deionized water, add 1 mL of 1 mol / L sodium hydroxide aqueous solution, stir to dissolve uniformly, and make up to 100 mL to prepare a 5 mg / mL hematin chloride solution (2-8℃ avoid light storage).

[0179] Inulin refers to SDF substances derived from chicory tubers, which are commercially available.

[0180] 2, Fecal sample collection and in vitro fermentation

[0181] 5-6 week-old healthy male C57BL / 6 clean level mice with uniform body weight were selected. The feeding temperature was 20℃±2℃, and the relative humidity was 55-60%. The mice were freely fed and watered during the adaptive feeding period after purchase, and after 1 week, the mice were randomly divided into 2 groups: normal mice (standard feed, normal saline) and obese mice (high-fat feed, normal saline), 7 in each group. The normal mice were fed with standard feed during the whole experiment, and the obese mice were fed with high-fat feed (Research Diets D124926 0kcal%Fat). The mice were fasted for 12 h without water, weighed and recorded the initial body weight of the mice. Fresh fecal samples of mice were collected when the body weight of mice was significantly different from the initial value after 14 weeks of feeding.

[0182] The fecal samples of normal mice were mixed and divided into normal control group (normal), probiotic group (PB), inulin group (positive), low-dose wheat sprout SDF group (SDF-L), high-dose wheat sprout SDF group (SDF-H), and wheat sprout IDF+low-dose wheat sprout SDF group (IDF+SDF-L).

[0183] The fecal samples of obese mice were mixed and divided into negative control group (negative), probiotic group (PB), inulin group (positive), low-dose wheat sprout SDF group (SDF-L), high-dose wheat sprout SDF group (SDF-H), and wheat sprout IDF+low-dose wheat sprout SDF group (IDF+SDF-L).

[0184] Each group of fresh fecal samples was added to sterile 0.1 mol / L phosphate buffer (pH 6.8) to prepare a 10 g / 100 mL fecal suspension for inoculation into prepared GAM basal medium.

[0185] In normal mice, 500 μL of the fecal suspension was inoculated into 5 mL of GAM basal medium for the normal control group; 500 μL of the fecal suspension was inoculated into 5 mL of GAM basal medium containing 30 mg of probiotic bacteria (Lactobacillus plantarum) for the probiotic bacteria group; 500 μL of the fecal suspension was inoculated into 5 mL of GAM basal medium containing 60 mg of inulin for the inulin group; 500 μL of the fecal suspension was inoculated into 5 mL of GAM basal medium containing 30 mg or 60 mg of wheat sprout SDF prepared according to the method of Example 3 for the low- and high-dose wheat sprout SDF groups, respectively; 500 μL of the fecal suspension was inoculated into 5 mL of GAM basal medium containing 70 mg of wheat sprout IDF and 30 mg of wheat sprout SDF prepared according to the method of Example 3 for the wheat sprout IDF + low-dose SDF group. The medium of each group except the probiotic bacteria group was first added to an anaerobic tube and sterilized at 115°C for 30 min, and then the fecal suspension was added. The medium of the probiotic bacteria group was first sterilized at 115°C for 30 min, and then the probiotic bacteria and the fecal suspension were added. The culture solution of each group was sampled at 0, 16, 24, and 48 h of incubation at 37°C to measure the pH value. The results are shown in Table 1. Figure 13 .

[0186] In obese mice, 500 μL of the fecal suspension was inoculated into 5 mL of GAM basal medium for the negative control group; 500 μL of the fecal suspension was inoculated into 5 mL of GAM basal medium containing 30 mg of probiotic bacteria (Lactobacillus plantarum) for the probiotic bacteria group; 500 μL of the fecal suspension was inoculated into 5 mL of GAM basal medium containing 60 mg of inulin for the inulin group; 500 μL of the fecal suspension was inoculated into 5 mL of GAM basal medium containing 30 mg or 60 mg of wheat sprout SDF prepared according to the method of Example 3 for the low- and high-dose wheat sprout SDF groups, respectively; 500 μL of the fecal suspension was inoculated into 5 mL of GAM basal medium containing 70 mg of wheat sprout IDF and 30 mg of wheat sprout SDF prepared according to the method of Example 3 for the wheat sprout IDF + low-dose SDF group. The medium of each group except the probiotic bacteria group was first added to an anaerobic tube and sterilized at 115°C for 30 min, and then the fecal suspension was added. The medium of the probiotic bacteria group was first sterilized at 115°C for 30 min, and then the probiotic bacteria and the fecal suspension were added. The culture solution of each group was sampled at 0, 16, 24, and 48 h of incubation at 37°C to measure the pH value. The results are shown in Table 2. Figure 14 .

[0187] 3. Changes in pH value after intestinal flora fermentation of wheat sprout SDF / IDF

[0188] The dynamic changes of pH value of wheat sprout SDF / IDF fermented by intestinal flora of normal mice and obese mice at different time points are shown in Table 1 Figure 13 With Figure 14 .

[0189] As Figure 13 , for normal mice, the pH of fermentation broth of each group was between 7-8 at 0h, that is, it was weakly alkaline. At 16h of fermentation, the pH of fermentation broth of each group was below 6, that is, it was weakly acidic; among them, the pH of fermentation broth of high-dose wheat sprout SDF group was significantly lower than that of the normal control group (p<0.05), and there was no difference between the low-dose wheat sprout SDF group, the probiotic group, the inulin group, the IDF+SDF group and the normal control group (p>0.05), indicating that high-dose wheat sprout SDF can effectively promote intestinal flora to produce acid. At 24h of fermentation, the pH of the normal control group did not change, and the pH of the inulin group slightly increased; the pH of the probiotic group, the low-dose wheat sprout SDF group and the IDF+SDF group further decreased. At this time, the pH of the fermentation broth of all experimental groups (including high and low-dose wheat sprout SDF groups, inulin group, IDF+low-dose SDF group and probiotic group) was significantly lower than that of the normal control group (p<0.05). At 48h of fermentation, except that the pH of the normal control and the IDF+low-dose SDF group did not change, the pH of the other groups showed an upward trend, but the pH was below 5.5. Among them, the pH of the fermentation broth of the high-dose wheat sprout SDF group, the probiotic group and the IDF+low-dose SDF group was significantly lower than that of the normal control group (p<0.05). It can be seen that high and low-dose wheat sprout SDF, probiotics and IDF+SDF can be fermented to produce acid to reduce the pH to 4.3-4.8 at 24h of fermentation. Continue to extend the fermentation time to 48h, each sample will not make the fermentation broth produce excessive acid, and the pH of the fermentation broth will rise to 4.8-5.4.

[0190] As Figure 14For obese mice, the pH of the fermentation broth of each group was between 7 and 8 at 0 h, i.e., all were weakly alkaline, and there was no significant difference between groups (p > 0.05). At 16 h of fermentation, the pH of the fermentation broth of each group was below 6, and there was a significant difference compared with the negative control group (p < 0.05); among them, the pH of the fermentation broth of the IDF+SDF group was the lowest (pH 4.5 ± 0.1), and the pH of the fermentation broth of the high and low dose wheat sprout SDF groups and the probiotic group was between 5.4 and 5.6. At 24 h of fermentation, the pH of the fermentation broth of the negative control group showed a downward trend, and the pH of the fermentation broth of the other sample groups was similar to that at 16 h and was significantly different compared with the negative control group (p < 0.05). At 48 h of fermentation, the pH of the fermentation broth of the negative control group further decreased, and the pH of the fermentation broth of the low dose wheat sprout SDF and IDF+SDF groups slightly increased, and the pH of the fermentation broth of the other groups did not change. The pH of the fermentation broth of the negative control group did not decrease significantly in the early stage of fermentation, but decreased continuously in the later stage. It was speculated that there were two reasons for this. One was that the intestinal bacteria stored intracellular polysaccharides such as glycogen when nutrients were sufficient, and decomposed them into organic acids such as lactic acid when exogenous sugar was lacking. The other was that the intestinal bacteria (such as Clostridium) decomposed proteins or amino acids, and generated byproducts such as acetic acid / propionic acid and ammonia, hydrogen sulfide through deamination. The potential risk was that excessive fermentation of proteins would increase harmful substances such as ammonia and hydrogen sulfide, which might damage intestinal epithelial cells.

[0191] In summary, high and low dose wheat sprout SDF, probiotics, and IDF+low dose SDF can be effectively degraded by intestinal flora into monosaccharides or oligosaccharides through the secretion of polysaccharide-degrading enzymes, and then through anaerobic metabolic pathways such as glycolysis to produce organic acids (such as lactic acid, pyruvic acid), and further converted into short-chain fatty acids (SCFAs, including acetic acid, propionic acid, butyric acid). Among them, wheat sprout SDF and probiotics can make normal mouse intestinal flora rapidly ferment to produce SCFAs, and wheat sprout IDF+SDF can make obese mouse intestinal flora rapidly ferment to produce SCFAs. Since the intestinal flora of obese mice has already been disturbed, an appropriate proportion of IDF and SDF (7:3) is more beneficial to the fermentation of intestinal flora to produce SCFAs than pure SDF. The metabolism of SDF / IDF by intestinal flora to produce SCFAs has the following benefits: ① SCFAs are the main energy source for colon epithelial cells (70% of energy is provided by butyric acid), and can enhance intestinal barrier function and reduce harmful substances entering the blood. ② SCFAs participate in the regulation of immune response and the inhibition of inflammation by activating G protein-coupled receptors and inhibiting histone deacetylase, and have potential improvement effects on metabolic diseases such as obesity and diabetes. ③ It can enrich beneficial bacteria and inhibit the growth of pathogenic bacteria, and optimize the intestinal microecology. However, as the fermentation time is prolonged, wheat sprout SDF, IDF, and probiotics will not excessively ferment to produce acid, causing the pH of the intestine to be too low, leading to acid-base imbalance or intestinal dysfunction.

[0192] 4. Effect of wheat sprout SDF / IDF on intestinal flora of normal mice

[0193] Step 2: After 48h of culture, samples were taken from each group of normal mice. The 16S rDNA sequencing technology was used to analyze the colon contents of mice, and the intestinal flora was systematically analyzed at the door and genus level. The differences in intestinal flora of mice in different groups were analyzed to reveal the internal mechanism of wheat sprout SDF / IDF in regulating intestinal microecology.

[0194] The library concentration should be above 2nM after evaluation by Agilent 2100 Bioanalyzer and Illumina library quantification kit. The qualified sequencing library was gradient diluted, mixed according to the required sequencing amount, and denatured to single strand by NaOH. The NovaSeq 6000 sequencer was used for 2x250bp double-end sequencing, and the corresponding reagent was NovaSeq6000SP Reagent Kit(500cycles). According to the species abundance table and species annotation table, the TOP30 species classification was selected, and the relative abundance of each group was displayed in different forms. The results are shown in Figure 15 、 16 and 17.

[0195] As shown in Figure 15 Bray-Curtis similarity analysis (ANOSIM) in Figure A, significant differences were found between different treatment groups (p<0.05), indicating that wheat sprout SDF / IDF intervention significantly changed the structure of intestinal flora in mice. As shown in Figure 15 Figure B, the stress value of non-metric multidimensional scaling analysis (NMDS) of wheat sprout SDF / IDF and probiotic PB was lower than the ideal threshold value of 0.1, which was 0.04944, verifying the reliability of the NMDS analysis results, ensuring that it could accurately reflect the similarity and difference of the flora structure between samples. From the NMDS graph, it can be clearly observed that the sample points of the normal control group (normal) showed a clear separation from other groups, indicating that the intestinal flora structure of mice under normal physiological conditions had unique clustering characteristics. The sample points of inulin group (positive-N, inulin as positive material), high-dose wheat sprout SDF group (SDF-H-N), low-dose wheat sprout SDF group (SDF-L-N), probiotic group (PB-N) and wheat sprout IDF-SDF group (IDF-SDF-N) each formed a specific aggregation area, indicating that wheat sprout SDF / SDF+IDF and probiotic PB treatment could both change the flora structure.

[0196] From Figure 16The heat map and relative abundance of the effects of wheat sprout SDF / IDF on the intestinal flora of mice at the door level showed that Bacteroidota (Bacteroidetes) and Bacillota (formerly known as Firmicutes) were the dominant phyla in the intestines of mice. The normal ratio of Bacteroidetes and Firmicutes is very important for maintaining intestinal health. In a generally healthy intestine, Bacteroidetes accounts for 16.9% to 32% of the total amount of intestinal flora, and Firmicutes accounts for 65% to 79.4%. The ratio of Firmicutes to Bacteroidetes (F / B ratio) is an important indicator of intestinal health. Changes in this ratio are often associated with intestinal flora imbalance and may be related to various diseases. The inulin group (positive-N, inulin as a positive substance), high-dose wheat sprout SDF group (SDF-H-N), low-dose wheat sprout SDF group (SDF-L-N), probiotic group (PB-N), and wheat sprout IDF-SDF group (IDF-SDF-N) all increased the abundance of Firmicutes in the intestines of mice. In particular, the wheat sprout IDF-SDF group, followed by the probiotic group. Firmicutes can inhibit the proliferation of harmful bacteria in the intestine and stimulate the immune system, suggesting that wheat sprout SDF / SDF+IDF and probiotics can both improve the intestinal flora state.

[0197] By Figure 17 The effects of wheat sprout SDF / IDF on the composition of intestinal flora at the genus level in mice showed that the abundance of Lactobacillus (Lactobacillus) in the Firmicutes phylum was the highest in the normal control group, followed by Bifidobacterium (Bifidobacterium) in the Actinobacteria phylum, and the two accounted for nearly 40% of the total. The inulin group (positive-N, inulin as a positive substance), high-dose wheat sprout SDF group (SDF-H-N), low-dose wheat sprout SDF group (SDF-L-N), probiotic group (PB-N), and wheat sprout IDF-SDF group (IDF-SDF-N) all increased the abundance of Lactobacillus in the intestines of mice. In particular, the wheat sprout IDF-SDF group, followed by the probiotic group. Lactobacillus can inhibit the growth of harmful bacteria, maintain intestinal flora balance, prevent intestinal infection and inflammation, break down cellulose and other indigestible substances in food, promote food digestion and nutrient absorption, enhance the function of the intestinal mucosal barrier to prevent harmful substances from entering the blood circulation, and improve immunity. It can regulate the immune system's response, reduce the symptoms of allergic reactions, lower cholesterol levels in the blood to prevent cardiovascular disease, and inhibit the growth of harmful bacteria in the oral cavity to prevent tooth decay and bad breath. In addition to the absolute dominance of Lactobacillus, Bifidobacterium and Enterococcus (Enterococcus) showed secondary dominant distribution characteristics in the intestinal flora of mice in each sample group. Both of these bacteria are probiotics in the intestine. Therefore, wheat sprout SDF / SDF+IDF at the genus level can improve the intestinal microecological environment and improve the health status of the host.

[0198] 5. Effects of wheat sprout SDF / IDF on gut microbiota in high-fat diet-induced obese mice

[0199] Following the method in step 4, after culturing the obese mice in each group for 48 hours in step 2, samples were taken for systematic analysis of the gut microbiota. The results are shown below. Figure 18 , 19 And 20.

[0200] like Figure 18 As shown in Figure A, the Bray-Curtis similarity analysis (ANOSIM) revealed significant differences between the different treatment groups (p = 0.001), indicating that wheat sprout SDF / IDF intervention significantly altered the gut microbiota structure in mice. The non-metric multidimensional scaling (NMDS) analysis of wheat sprout SDF / IDF showed stress values ​​below the ideal threshold of 0.1 (0.05357), validating the reliability of the NMDS analysis results and ensuring its accurate reflection of the similarity and differences in microbiota structure among samples. The NMDS plot clearly shows that the negative control group exhibited a significant separation from other groups, indicating that each group of samples contributed unique clustering characteristics to the mouse gut microbiota structure. In the inulin group (positive-D, with inulin as the positive substance), the high-dose wheat sprout SDF group (SDF-HD), the low-dose wheat sprout SDF group (SDF-LD), the probiotic group (PB-D), and the wheat sprout IDF-SDF group (IDF-SDF-D), each sample point formed a specific aggregation region, suggesting that wheat sprout SDF / SDF+IDF intervention can promote changes in the microbial community structure.

[0201] Depend on Figure 19The heatmap of the effects of wheat sprout SDF / IDF on the intestinal flora of mice at the door level shows that the abundance of Pseudomonadota (Pseudomonas) is the highest in the negative control group, accounting for nearly 60%; followed by Firmicutes, Actinobacteria, and Bacteroidetes. In the intestinal flora of the negative control group, the relative abundance of Firmicutes and Bacteroidetes is significantly lower than the normal level (less than 30%), far from the benchmark proportion of more than 80% in a healthy state. In addition, Pseudomonas, which has the highest proportion, is a conditional pathogen and may cause disease under certain circumstances (such as low immunity). The above shows that long-term (12 weeks) high-fat feed can cause significant disorder of the intestinal flora of mice. The low-dose wheat sprout SDF group (SDF-L-D), the probiotic group (PB-D), and the wheat sprout IDF-SDF group (IDF-SDF-D) can all reduce the abundance of Pseudomonas in the intestinal flora of mice, especially the wheat sprout IDF-SDF group, which has the most obvious decrease in the abundance of Pseudomonas, less than 10%. Compared with the negative control group, the wheat sprout IDF-SDF group, the high-dose wheat sprout SDF group, the probiotic group, and the inulin group can all increase the abundance of Firmicutes in the intestinal flora of mice, especially the wheat sprout IDF-SDF group, which has the largest increase in the abundance of Firmicutes, indicating that wheat sprout SDF / SDF+IDF can improve the imbalance of the intestinal flora of obese mice induced by high-fat feed.

[0202] By Figure 20 The effects of wheat sprout SDF / SDF+IDF on the composition of intestinal flora of mice at the genus level show that the abundance of Escherichia-Shigella (Escherichia-Shigella) is the highest in the negative control group, accounting for nearly 60%; the abundance of Lactobacillus is low, less than 3%. Among them, Escherichia coli is a common bacterium in the intestinal tract of humans and animals, most strains are harmless, but some pathogenic strains may cause intestinal or extra-intestinal infections, leading to diseases such as diarrhea, urinary tract infection, and sepsis. Shigella is a pathogenic bacterium that can cause acute gastroenteritis (bacterial dysentery) after infection, leading to intestinal tissue damage and necrosis.

[0203] The low-dose wheat sprout SDF group (SDF-L-D), the probiotic group (PB-D), and the wheat sprout IDF-SDF group (IDF-SDF-D) can all reduce the abundance of Escherichia-Shigella in the intestinal tract of mice, and the wheat sprout IDF-SDF group is the most obvious, with an abundance of less than 10%. Compared with the negative control group, the wheat sprout IDF-SDF group, the high-dose wheat sprout SDF group, the probiotic group, and the inulin group can all increase the abundance of Lactobacillus in the intestinal tract of mice, and the wheat sprout IDF-SDF group has the largest increase, with an abundance of nearly 60%. Therefore, at the genus level, the wheat sprout SDF / SDF+IDF can improve the disturbed intestinal microecological environment of high-fat diet-induced obese mice and improve the health status of the host.

[0204] Example 7: Determination of physicochemical properties of insoluble dietary fiber (IDF) of wheat sprouts

[0205] According to the method of step (3) of Example 3, the precipitate was washed with water, dried, crushed, and sieved (>60 mesh, 60-100 mesh, 100-150 mesh, 150-200 mesh, and 200-300 mesh) to obtain wheat sprout IDF powder with different particle sizes.

[0206] In addition, fresh wheat sprout raw material was dried at 65°C to a water content of less than 11%, crushed, and sieved (>60 mesh, 60-100 mesh, 100-150 mesh, 150-200 mesh, and 200-300 mesh) to obtain wheat sprout powder with different particle sizes.

[0207] 1. Hydration capacity

[0208] 1.00 g (M1) of the sample was placed in a 100 mL beaker, 50 mL of deionized water was added, and it was left to stand at 37°C for 2 h. The sample residue was filtered with a 0.71 mm nylon mesh until no water droplets were observed, and the wet weight M2 of the residue was measured. The sample was then dried in an oven, and the dry weight M3 was measured. The water holding capacity was (M2-M1) / M1, and the unit was g / g. The bound water capacity was (M2-M3) / M3, and the unit was g / g.

[0209] The water holding capacity and bound water capacity of the wheat sprout raw material and the wheat sprout IDF are shown in Table 1. Figure 21 Compared with Figure 22 .

[0210] The chemical structure of wheat sprout dietary fiber contains a large number of hydrophilic groups, which has good hydration capacity. This physicochemical property enables it to absorb water and prevent various intestinal diseases. The hydration properties of wheat sprout dietary fiber are related to its own chemical structure characteristics, the porosity of the fiber, the particle size, the temperature, the pH, the type of ions, and the ionic strength.

[0211] From the aboveFigure 21 It can be seen that the particle size has little effect on the water holding capacity of wheat sprout raw material under the same conditions, and the water holding capacity of different particle sizes of wheat sprout raw material is between 4.9 g / g and 6.9 g / g, and there is no difference between different particle sizes (p>0.05). The particle size has a great effect on the water holding capacity of IDF of wheat sprout under the same conditions, and with the decrease of particle size, the water holding capacity of IDF of wheat sprout appears a significant downward trend and then a significant upward trend, and the water holding capacity of IDF of different particle sizes of wheat sprout is between 6.3 g / g and 9.0 g / g, and the smaller the particle size of IDF of wheat sprout, the better the water holding capacity. It can be seen that the water holding capacity of IDF of wheat sprout is better than that of wheat sprout raw material.

[0212] Figure 22 It can be seen that the particle size has little effect on the water holding capacity of wheat sprout raw material under the same conditions, and the water holding capacity of different particle sizes of wheat sprout raw material is between 4.9 g / g and 6.9 g / g, and there is no difference between different particle sizes (p>0.05). The particle size has a great effect on the water holding capacity of IDF of wheat sprout under the same conditions, and with the decrease of particle size, the water holding capacity of IDF of wheat sprout appears a significant downward trend and then a significant upward trend, and the water holding capacity of IDF of different particle sizes of wheat sprout is between 6.3 g / g and 9.0 g / g, and the smaller the particle size of IDF of wheat sprout, the better the water holding capacity. It can be seen that the water holding capacity of IDF of wheat sprout is better than that of wheat sprout raw material.

[0213] 2. Oil holding capacity

[0214] Take 1.00 g (M1) of sample and place it in a 50 mL centrifuge tube, add 25 g of soybean oil, and stand at 37°C for 1 h, centrifuge at 3000 r / min for 20 min, pour off the upper oil, and then filter the remaining part with a filter screen, and then use filter paper to absorb the excess oil and weigh M2. Oil holding capacity = (M2-M1) / M1, unit g / g.

[0215] The oil holding capacity of wheat sprout raw material and IDF of wheat sprout is shown in Table 4. Figure 23

[0216] The oil holding capacity is mainly related to the surface properties and particle size of wheat sprout dietary fiber, and may also be related to hydration properties, total charge density, etc. From the results of the experiment, it can be seen that the oil holding capacity of wheat sprout raw material is better than that of IDF of wheat sprout, and the oil holding capacity of IDF of wheat sprout is better than that of IDF of wheat sprout. Figure 23 ​It was found that the oil holding capacity of wheat sprout was greatly affected by the particle size. With the decrease of the particle size, the oil holding capacity of wheat sprout decreased significantly. The oil holding capacity of wheat sprout with different particle sizes was between 0.9 g / g and 2.2 g / g. The particle size greatly affected the oil holding capacity of IDF of wheat sprout. With the decrease of the particle size, the oil holding capacity of IDF of wheat sprout decreased significantly. The effect of the particle size on the oil holding capacity of IDF of wheat sprout was the same as that of the wheat sprout, i.e. the larger the particle size, the stronger the oil holding capacity. This might be because when the particle size gradually decreased, the surface area gradually increased, the hydrophilic groups in the molecules of IDF of wheat sprout were exposed, and the aggregation phenomenon occurred, thus the oil adsorption capacity gradually decreased. The oil holding capacity of IDF of wheat sprout with different particle sizes was between 0.1 g / g and 3.3 g / g. It was found that the oil holding capacity of IDF of wheat sprout was better than that of the wheat sprout when the particle size was large, and vice versa.

[0217] 3. Sodium cholate adsorption capacity

[0218] Cholate refers to a kind of amphiphilic macromolecule with steroidal nucleus structure from human and animal bile. It is believed that the adsorption of bile acid by dietary fiber is one of the mechanisms of its blood lipid-lowering function. Studies have shown that dietary fiber promotes the metabolism of cholesterol by adsorbing cholate and expelling it out of the body, reduces the reabsorption of bile acid, and breaks the enterohepatic circulation of cholesterol, so that the level of cholesterol in the body is significantly reduced. Therefore, understanding the ability of dietary fiber to adsorb cholate can help understand its ability to regulate blood lipids.

[0219] Sodium cholate standard curve: accurately weigh 0, 10, 20, 40, 60, 80, 100 mg of sodium cholate and place them in 50 mL volumetric flasks, dissolve with deionized water and dilute to volume. Take 1 mL of each different concentration of sodium cholate standard solution into a 15 mL test tube with a stopper, add 6 mL of 45% H2SO4, mix well, then add 1 mL of 0.3% volume concentration of furfural aqueous solution, react in a 65°C constant temperature water bath for 30 min, cool to room temperature, and measure the absorbance at 620 nm. Draw the standard curve with the concentration of sodium cholate as the abscissa and the absorbance value as the ordinate. Figure 24 The linear regression equation of the sodium cholate standard curve is: y = 0.2469x + 0.0503, R 2 = 0.9945.

[0220] Take 100 mL of 0.15 mol / L NaCl aqueous solution containing 0.2 g of sodium cholate into a 250 mL conical flask, add 0.5 g of sample into the conical flask and stir, centrifuge for 20 min in a constant temperature shaking incubator at 37°C for 2 h, accurately transfer 1 mL of supernatant into a 15 mL test tube with a stopper, add 6 mL of 45% H2SO4, mix, then add 1 mL of 0.3% aqueous furfural solution, react in a 65°C constant temperature water bath for 30 min, cool to room temperature, then measure the absorbance at 620 nm, and calculate the content of sodium cholate according to the sodium cholate standard curve, and calculate the adsorption rate of sodium cholate according to the content difference before and after the reaction.

[0221] The adsorption of cholate salt by wheat sprout raw material and wheat sprout IDF is shown in Table 2. Figure 24 The particle size has a great influence on the adsorption capacity of sodium cholate by wheat sprout raw material. As the particle size decreases from less than 60 mesh to 60-100 mesh, the adsorption capacity of sodium cholate by wheat sprout raw material decreases significantly from more than 50% to less than 30%; when the particle size continues to decrease, the adsorption capacity of sodium cholate by wheat sprout raw material remains basically unchanged; when the particle size continues to decrease to 200-300 mesh, the adsorption capacity of sodium cholate slightly increases but does not reach significance. The particle size has a great influence on the adsorption capacity of sodium cholate by wheat sprout IDF, and as the particle size continuously decreases, the adsorption capacity of sodium cholate by wheat sprout IDF shows a fluctuating trend of significant increase, significant decrease and significant increase again. When the particle size is 200-300 mesh, the adsorption capacity of sodium cholate by wheat sprout IDF is the strongest, reaching 25.43%. Overall, the adsorption capacity of sodium cholate by wheat sprout raw material is stronger than that by wheat sprout IDF.

[0222] 4. Cholesterol adsorption capacity

[0223] Adsorption performance refers to the adsorption of organic matter (such as cholesterol, oil, bile acid, etc.) and the ability to promote excretion. Cholesterol is an important component of biomolecular cell membranes, and its normal content in the human body is about 240 g. If the cholesterol metabolism of the human body is impaired, the increase in the content of cholesterol in the blood causes local deposition of cholesterol in the blood vessels, which further develops into atherosclerosis. The concentration of cholesterol in the blood serum is closely related to various cardiovascular diseases. High cholesterol blood disease caused by cholesterol is one of the most popular and most harmful chronic non-communicable diseases in the world, and is also one of the most difficult and difficult diseases in current clinical practice. The adsorption capacity of cholesterol by wheat sprout raw material and wheat sprout IDF under the conditions of pH 2.0 (simulating the environment of human gastric juice) and pH 7.0 (simulating the environment of human small intestine) was detected respectively.

[0224] Cholesterol standard curve: 0.00 mL, 0.05 mL, 0.10 mL, 0.15 mL, 0.20 mL, 0.25 mL, 0.30 mL and 0.35 mL of 0.100 mg / mL cholesterol standard working solution prepared with anhydrous ethanol were respectively taken in 10 mL test tubes, 0.20 mL of o-phthalaldehyde aqueous solution (1 mg / mL) and 4 mL of mixed acid (glacial acetic acid: concentrated sulfuric acid = 1:1, by volume) were added along the tube wall to make the total volume reach 0.40 mL in each tube, and then mixed uniformly. After standing at room temperature for 10 min, the absorbance was measured at 550 nm. The standard curve of cholesterol was plotted with the amount of cholesterol (mg) as the abscissa and the absorbance as the ordinate. Figure 25 The linear regression equation of the cholesterol standard curve was y = 19.803x + 0.081, R 2 = 0.9968.

[0225] 1.000 g of sample was weighed into a 100 mL conical flask, 50 mL of egg yolk solution (fresh egg yolk was fully stirred with 9 times the mass of distilled water to obtain the egg yolk solution) was added, the solution was adjusted to pH 2.0 and 7.0, respectively, and then shaken on a shaking bed (temperature 37°C, rotation speed 120 r / min) for 2 h. After centrifugation at 6000 r / min for 20 min, 1 mL of supernatant was taken and diluted 5 times with 90% acetic acid. Sample group: 0.1 mL of the diluted solution was taken, 0.3 mL of glacial acetic acid was added, 0.20 mL of o-phthalaldehyde aqueous solution (1 mg / mL) and 4 mL of mixed acid (glacial acetic acid: concentrated sulfuric acid = 1:1) were added along the tube wall, and then mixed uniformly. After standing at room temperature for 10 min, the absorbance was measured at 550 nm. The cholesterol content in the supernatant after adsorption was obtained according to the cholesterol standard curve.

[0226] Another 0.1 mL of egg yolk solution was taken, 0.3 mL of glacial acetic acid was added, 0.20 mL of anhydrous ethanol and 4 mL of mixed acid (glacial acetic acid: concentrated sulfuric acid = 1:1) were added along the tube wall, and then mixed uniformly. After standing at room temperature for 10 min, the absorbance was measured at 550 nm. The cholesterol content in the egg yolk solution was obtained according to the cholesterol standard curve.

[0227] The cholesterol adsorption capacity = (the amount of cholesterol in the egg yolk solution - the amount of cholesterol in the supernatant after adsorption) / the mass of the sample, unit mg / g.

[0228] Figure 25The results showed that the size of the particle had a great influence on the adsorption capacity of cholesterol of wheat sprout raw material. With the size of the particle decreasing, the adsorption capacity of cholesterol of wheat sprout raw material showed a fluctuation trend of significant decline, significant increase and significant decline again. When the size of the particle was less than 60 mesh, the adsorption capacity of cholesterol of wheat sprout raw material was the strongest, reaching 9.94 mg / g. The size of the particle had a great influence on the adsorption capacity of cholesterol of wheat sprout IDF. With the size of the particle decreasing, the adsorption capacity of cholesterol of wheat sprout IDF showed a trend of significant decline and slight increase. Like the wheat sprout raw material, when the size of the particle was less than 60 mesh, the adsorption capacity of cholesterol of wheat sprout IDF was the strongest, reaching 10.20 mg / g, which was slightly higher than that of the wheat sprout raw material.

[0229] Figure 26 The results showed that with the size of the particle decreasing from less than 60 mesh to 150-200 mesh, the adsorption capacity of cholesterol of wheat sprout raw material showed a fluctuation trend, but there was no significant difference. When the size of the particle continued to decrease to 200-300 mesh, the adsorption capacity of cholesterol decreased significantly. When the size of the particle was 100-150 mesh, the adsorption capacity of cholesterol of wheat sprout raw material was the strongest, reaching 10.90 mg / g. The size of the particle had a great influence on the adsorption capacity of cholesterol of wheat sprout IDF. With the size of the particle decreasing, the adsorption capacity of cholesterol of wheat sprout IDF showed a significant decline trend. Like the adsorption at pH 2.0, when the size of the particle was less than 60 mesh, the adsorption capacity of cholesterol of wheat sprout IDF was the strongest, reaching 20.12 mg / g, which was significantly higher than that of the wheat sprout raw material.

[0230] In summary, in the pH 2 and pH 7 environments, the wheat sprout raw material and the wheat sprout IDF basically showed a trend that the larger the size of the particle, the stronger the adsorption capacity of cholesterol. Among them, the adsorption capacity of cholesterol of the wheat sprout IDF in the pH 2 and pH 7 environments (the size of the particle was less than 60 mesh) was greater than that of the wheat sprout raw material, especially in the pH 7 environment, the adsorption capacity of cholesterol of the wheat sprout IDF was significantly better than that of the wheat sprout raw material, and was better than that in the pH 2 environment, indicating that the adsorption capacity of cholesterol of the wheat sprout IDF in the intestine was greater than that in the stomach. Liver and intestinal mucosa are the main places for the synthesis of cholesterol, and the adsorption characteristics of cholesterol of the wheat sprout IDF have certain physiological significance.

[0231] Comparative Example 1, direct hot water extraction

[0232] The method of Example 3 was used without adding hydrogen peroxide and cellulase, i.e. 2 g of wheat sprout powder was added to 80 mL of deionized water at a material-to-liquid ratio of 1 g:40 mL, and the mixture was placed in a constant-temperature oscillator, the parameters of which were adjusted to 40°C and 200 rpm, and the mixture was extracted by reciprocating rotary oscillation for 70 min. The mixture was centrifuged at 8000 r / min for 20 min, and 1.52 g of IDF with a mesh number of 200-300 was obtained from the precipitate according to the method of Example 3. 1.0 mL of the supernatant was taken, and the SDF content in the supernatant was detected by the phenol-sulfuric acid method, and the yield was converted. The water-holding capacity, bound water capacity and cholesterol adsorption capacity of the IDF were detected according to the method of Example 7. The results are shown in Table 4.

[0233] Table 4 Influence of different extraction processes on the yield of SDF of wheat sprout and the physicochemical properties of IDF

[0234]

[0235] As can be seen from Table 4, different extraction processes have a great influence on the yield of SDF of wheat sprout, and the extraction process of enzyme and hydrogen peroxide can significantly improve the yield of SDF of wheat sprout, which is 3.488 times higher than that of traditional hot water extraction. The water-holding capacity of the IDF is 2.944 times higher than that of the traditional hot water extraction. The bound water capacity of the IDF is 1.078 times higher than that of the traditional hot water extraction. The cholesterol adsorption capacity of the IDF is 1.324 times higher than that of the traditional hot water extraction. The hydroxyl radical and ABTS + scavenging rates are similar.

[0236] Example 2, influence of cellulase extraction on the yield of SDF of wheat sprout

[0237] The method of Example 3 was used without adding hydrogen peroxide, i.e. 2 g of wheat sprout powder was added to 80 mL of deionized water at a material-to-liquid ratio of 1 g:40 mL, and the mixture was placed in a constant-temperature oscillator, the parameters of which were adjusted to 40°C and 200 rpm, and the mixture was extracted by reciprocating rotary oscillation for 70 min. The mixture was centrifuged at 8000 r / min for 20 min, and 1.52 g of IDF with a mesh number of 200-300 was obtained from the precipitate according to the method of Example 3. 1.0 mL of the supernatant was taken, and the SDF content in the supernatant was detected by the phenol-sulfuric acid method, and the yield was converted. The water-holding capacity, bound water capacity and cholesterol adsorption capacity of the IDF were detected according to the method of Example 7. The results are shown in Table 4.

[0238] Table 5 Influence of different extraction processes on the yield of SDF of wheat sprout

[0239]

[0240] As can be seen from Table 5, different extraction processes have a great influence on the yield of SDF of wheat sprout, and the extraction process of enzyme and hydrogen peroxide can significantly improve the yield of SDF of wheat sprout, which is 3.488 times higher than that of traditional hot water extraction. The water-holding capacity of the IDF is 2.944 times higher than that of the traditional hot water extraction. The bound water capacity of the IDF is 1.078 times higher than that of the traditional hot water extraction. The cholesterol adsorption capacity of the IDF is 1.324 times higher than that of the traditional hot water extraction. The hydroxyl radical and ABTS + scavenging rates are similar.

[0241] Comparative Example 3: Effects of IDF + Inulin on Physicochemical Properties of Wheat Sprouts

[0242] Different particle sizes of wheat sprout IDF were prepared using the method in Example 3. The IDF was then uniformly mixed with inulin at a mass ratio of 1:1. The water-holding capacity, water-binding capacity, oil-holding capacity, and cholesterol adsorption capacity were tested using the method in Example 7. The results are shown in [Figure 7]. Figure 27 .

[0243] Depend on Figure 27 It was found that the physicochemical properties of the extracted wheat germ IDF, after being uniformly mixed with inulin in a 1:1 ratio, differed from those of wheat germ IDF alone. The water-holding and bound water capacities of wheat germ IDF + inulin differed from those of wheat germ IDF alone; both were higher at larger particle sizes (mesh size > 60), reaching 10.07 g / g and 9.75 g / g, respectively. The oil-holding capacity also differed from that of wheat germ IDF alone, being higher at a mesh size of 100-150, at 1.12 g / g. The cholesterol adsorption capacity was exactly the opposite of the hydration characteristics (water-holding and bound water capacity); the adsorption capacity of wheat germ IDF + inulin was highest at smaller particle sizes (200-300 mesh), with adsorption capacities of 16.01 mg / g at pH 2.0 and 22.61 mg / g at pH 7.0.

[0244] Example 8

[0245] Add 1g of wheat sprout powder to 0.004g of cellulase. Add 0.8mL of 30% hydrogen peroxide and 39.2mL of deionized water at a wheat sprout powder to wheat sprout powder liquid-to-solid ratio of 40:1 (mL / g), bringing the final hydrogen peroxide concentration to 0.6%. Place the mixture in a constant-temperature shaker and shake at 100℃ and 200rpm for 20 minutes. Centrifuge (8000r / min for 20 minutes) to obtain the supernatant. Take 1.0mL of the supernatant and determine the SDF content using the phenol-sulfuric acid method. The extraction yield, calculated according to Example 1, is 10.27%.

[0246] Example 9

[0247] 1 g wheat sprout powder was added to 0.012 g cellulase, 0.8 mL of 30% hydrogen peroxide and 39.2 mL of deionized water were added according to the liquid material ratio of 40:1 (mL / g) of wheat sprout powder, so that the final concentration of hydrogen peroxide was 0.6%, and it was placed in a constant temperature oscillator. The parameters of the oscillator were adjusted to 40°C, 200 rpm, reciprocating rotary oscillation for 100 min. After taking out, centrifugation (8000 r / min for 20 min) was performed to obtain the supernatant. 1.0 mL of the supernatant was taken, and the SDF content in the supernatant was detected by the phenol-sulfuric acid method, which was converted into the extraction yield according to Example 1, which was 11.62%.

[0248] Example 10

[0249] 1 g wheat sprout powder was added to 0.008 g cellulase, 0.1 mL of 30% hydrogen peroxide and 29.9 mL of deionized water were added according to the liquid material ratio of 30:1 (mL / g) of wheat sprout powder, so that the final concentration of hydrogen peroxide was 0.1%, and it was placed in a constant temperature oscillator. The parameters of the oscillator were adjusted to 100°C, 200 rpm, reciprocating rotary oscillation for 60 min. After taking out, centrifugation (8000 r / min for 20 min) was performed to obtain the supernatant. 1.0 mL of the supernatant was taken, and the SDF content in the supernatant was detected by the phenol-sulfuric acid method, which was converted into the extraction yield according to Example 1, which was 10.39%.

[0250] Example 11

[0251] 1 g wheat sprout powder was added to 0.004 g cellulase, 2 mL of 30% hydrogen peroxide and 48 mL of deionized water were added according to the liquid material ratio of 50:1 (mL / g) of wheat sprout powder, so that the final concentration of hydrogen peroxide was 1.2%, and it was placed in a constant temperature oscillator. The parameters of the oscillator were adjusted to 90°C, 200 rpm, reciprocating rotary oscillation for 60 min. After taking out, centrifugation (8000 r / min for 20 min) was performed to obtain the supernatant. 1.0 mL of the supernatant was taken, and the SDF content in the supernatant was detected by the phenol-sulfuric acid method, which was converted into the extraction yield according to Example 1, which was 11.41%.

[0252] The SDF of wheat sprout prepared in Examples 8-11 was subjected to ABTS and hydroxyl radical detection, worm survival rate detection under oxidative stress, and mouse intestinal flora metabolite detection by the methods of Examples 5 and 6. The results showed that all of them had strong free radical scavenging ability, increased the resistance of worms under oxidative stress, and could improve the imbalance of intestinal flora of obese mice induced by high-fat feed, and the IDF after extracting SDF had good hydration capacity and adsorption capacity of sodium cholate and cholesterol.

Claims

1. A wheat sprout functional dietary fiber, characterized by, The wheat sprout functional dietary fiber includes soluble dietary fiber and insoluble dietary fiber, and the main components of the soluble dietary fiber include guluronic acid, mannose, ribose, galacturonic acid, galactosamine, glucose, galactose, xylose, arabinose and fucose.

2. The wheat sprout functional dietary fiber according to claim 1, wherein The molar ratio of guluronic acid, mannose, ribose, galacturonic acid, galactosamine, glucose, galactose, xylose, arabinose and fucose in the soluble dietary fiber is 3.00:1.08:1.20:4.08:4.90:4.15:1.00:1.67:4.60:2.

04.

3. A method for preparing the functional dietary fiber of wheat sprout according to claim 1, characterized by, The method comprises: (1) adding hydrogen peroxide and deionized water in the wheat sprout powder and cellulase, and placing in a constant temperature oscillator for oscillation, adjusting the oscillator parameters to 40-100 DEG C, 200 rpm, reciprocating rotary oscillation extraction for 20-120 min; after the extraction is completed, it is placed in a 100 DEG C boiling water bath for 3 min, and centrifuged at 8000 rpm for 20 min to obtain a precipitate and a supernatant; (2) taking the supernatant of step (1), concentrating to one fifth of the original volume at 60 DEG C, then slowly adding the concentrate to 95% ethanol at a constant speed, and standing at 4 DEG C for 12 h, centrifuging at 3000 rpm for 10 min, taking the precipitate, dissolving with deionized water, and then deproteinizing by the sevag method until there is no absorption peak at 280 nm, and then freeze-drying to obtain wheat sprout soluble dietary fiber; (3) taking the precipitate of step (1), washing with water, drying, crushing, and sieving to obtain wheat sprout insoluble dietary fiber.

4. The production method according to claim 3, wherein In step (1), the cellulase is added in an amount of 0.1-1.8% based on the mass of the wheat sprout powder; the hydrogen peroxide is added in the form of a 30% aqueous solution, and the final concentration is 0.1-2%; the total volume of the hydrogen peroxide and deionized water is 20-65 mL / g based on the mass of the wheat sprout.

5. The production method according to claim 3, wherein In step (2), the volume ratio of ethanol to concentrate is 4:

1.

6. The production method according to claim 3, wherein In step (3), the water washing refers to washing the precipitate with an appropriate amount of deionized water for 2-3 times to remove residual soluble impurities, and each time the washing needs to be fully stirred before being discarded; the drying refers to placing the washed wet precipitate on a culture dish and drying it in a 70 DEG C oven until the weight is constant; and the sieving refers to crushing the dried blocky material with a universal crusher and sieving through different mesh screens to obtain powders of different particle sizes.

7. Use of the wheat sprout functional dietary fiber of claim 1 in the preparation of an antioxidant product.

8. Use of the wheat sprout functional dietary fiber of claim 1 in the preparation of an anti-aging product.

9. Use of the wheat sprout functional dietary fiber of claim 1 in the preparation of an intestinal flora regulator.

10. Use of the wheat sprout functional dietary fiber of claim 1 as a food additive.

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